Genetic markers of schizophrenia

ABSTRACT

The invention includes method of determining if a subject has a genetic predisposition to clinically diagnosed schizophrenia (SZ), schizotypal personality disorder (SPD), and/or schizoaffective disorder (SD).

CLAIM OF PRIORITY

This application is a divisional application of U.S. patent applicationSer. No. 11/859,168, filed on Sep. 21, 2007, issued as U.S. Pat. No.8,067,160 B2 which claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/948,392, filed on Jul. 6, 2007. The entirecontents of each of these applications are hereby incorporated byreference.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant No. R01HD29888 awarded by the National Institutes of Health. The Government hascertain rights in the invention.

STATE SPONSORED RESEARCH OR DEVELOPMENT

This invention was made in part with Grant No. CIF-127-COM from theKentucky Science and Technology Corporation under Contract No.144-401-06 to the University of Louisville and by Kentucky Science andTechnology Corporation Research and Development Voucher Contract#145-402-26 to SureGene, LLC.

TECHNICAL FIELD

This invention relates to genetic markers of schizophrenia (SZ),schizotypal personality disorder (SPD), and/or schizoaffective disorder(SD), and methods of use thereof.

BACKGROUND

Normal variation for the personality trait of schizotypy may haveorigins that overlap the etiological factors that produce thepsychiatric disease schizophrenia. Although schizophrenia has beenwidely researched in many disciplines for decades, the causes of thiscomplex disease still remain elusive. For diseases where factorsinfluencing ordinary variation in the population are the same as thosethat are etiologically relevant to the diagnosis of disease, a largeunselected sample may contribute beneficially to research on many traitsinstead of just one. Furthermore, the use of quantitative measures insuch a sample provides the advantage of tapping into variation in thelow and middle ranges, not just the diagnostically significant high endsof trait distributions.

SUMMARY

A whole autosomal screen was conducted for quantitative trait loci(QTLs) influencing adult schizotypy as measured by the schizophreniascale of the MINNESOTA MULTIPHASIC PERSONALITY INVENTORY-2 (MMPI-2) test(Hathaway and McKinley, 1989, supra). Tests for linkage based on anonclinical sample of 1,065 sibling and dizygotic (DZ) twin pairsrevealed a significant linkage on chromosome 4p15.1 spanning D2S391 anda highly suggestive linkage on 22q13.33 between D22S526 and D22S1744.These results point to two chromosomal regions that are associated withthe etiology of schizophrenia and other psychiatric disorders.

TABLE A SNP Markers Used for TDT Analysis (NCBI Genome Build 36.2)Position Gene Name Chromosome Marker Mb Alleles PI4K2B 4p15.2 rs31354824.8465 C/T PI4K2B 4p15.2 rs313567 24.8631 C/T KCNIP4 4p15.31 rs644798220.3693 A/C KCNIP4 4p15.31 rs10016449 20.4178 C/T KCNIP4 4p15.31rs3765119 20.4611 C/T KCNIP4 4p15.31 rs1364836 20.9425 C/T CERK 22q13.31rs801720 45.4608 G/T CERK 22q13.31 rs135667 45.4624 C/G CERK 22q13.31rs135678 45.4725 C/T CERK 22q13.31 rs135693 45.4812 C/G CERK 22q13.31rs1548977 45.5093 A/G CERK 22q13.31 rs710123 45.5239 A/G SHANK3 22q13.3rs713692 49.4566 C/T SHANK3 22q13.3 rs9616915 49.4644 C/T SHANK3 22q13.3rs9616816 49.4704 A/G SHANK3 22q13.3 rs739365 49.4872 C/T SHANK3 22q13.3rs6010063 49.5038 A/G SHANK3 22q13.3 rs756638 49.5186 A/G

Single nucleotide polymorphism (SNP) markers in a number of genes(including SH3 and Multiple Ankyrin Repeat Domains 3 (SHANK3), KvChannel Interacting Protein 4 Gene (KCNIP4), Ceramide Kinase Gene(CERK), and Phosphatidylinositol 4-Kinase Type 2 Beta Gene (PI4K2B))were used to evaluate families from the NIMH Schizophrenia GeneticsInitiative. Based on the results, an association of each of these geneswith schizophrenia spectrum disorders was identified. Thus, theinvention includes methods of determining risk of developingschizophrenia (SZ), schizotypal personality disorder (SPD) orschizoaffective disorder (SD) as described herein.

In one aspect, the invention includes methods for obtaining informationregarding a subject's risk for developing SZ, SD or SPD. The methodsinclude obtaining a test haplotype associated with schizophrenia asdescribed herein. The methods can also include obtaining a samplecomprising genomic DNA (gDNA) from the subject, and determining theidentity, absence or presence of a test haplotype associated with SZ, SDor SPD as described herein. In some embodiments, the methods includeobtaining a test haplotype for the subject comprising at least one testmarker that is listed in Table A, or is within 1 linkage disequilibriumunit (1 LDU) of a marker listed in Table A, wherein the haplotypeprovides information regarding the subject's risk of developing SZ, SPD,or SD. In some embodiments, the test marker is a marker listed in one ormore of Table A, or a marker within 1 linkage disequilibrium unit (1LDU) or a D′>0.75 of a polymorphism described herein, e.g., markers in aregion of chromosome 4p or 22q, e.g., in 22q13, e.g., in 4p between andincluding SNPs rs313548 and rs313567 at the PI4K2B locus and/or betweenrs6447982 and rs1364836 at the KCNIP4 locus; and/or in 22q13, e.g.,between rs801720 and rs710123 at the CERK locus, and/or between rs713692and rs756638 at the SHANK3 locus.

In some embodiments, the test haplotype includes at least one markerlisted in Table A, e.g., two or more markers listed in Table A. In someembodiments, the test haplotype includes two or more markers from onegene, or from each gene if two or more genes are used. In someembodiments, the test haplotype includes at least two markers, each froma different gene listed in Table A.

In some embodiments, the test haplotype includes at least one markerlisted in Table A and provides information regarding a subject's risk ofdeveloping SZ, under a narrower (DSM III/DSMIV) disease definition.

In some embodiments, the test haplotype provides information regarding asubject's risk of having a particular endophenotype, and/or one or morespecific symptoms, e.g., hallucinations, paranoia, mania, depression,obsessive-compulsive symptoms, etc., as well as response or lack ofresponse to drugs and comorbidity for substance and alcohol abuse.

The methods described herein can include obtaining a haplotype thatincludes two or more, e.g., two, three, four, five, or six markers.

Additionally, the methods can include determining the presence orabsence of other markers known to be associated with SZ, SD or SPD,e.g., outside of a region identified herein. A number of other suchmarkers are known in the art, e.g., as described herein.

The subject can be a mammal, e.g., a primate, preferably a higherprimate, e.g., a human (e.g., a patient having, or at risk of, SZ, SD orSPD). In one embodiment, the subject is a patient having SZ, SD or SPD(e.g., a patient suffering from early, intermediate or aggressive SZ, SDor SPD). In some embodiments, the methods described herein are used toobtain information regarding a subject's risk of developing SZ, SD orSPD, wherein the disorder is other than catatonic schizophrenia. In someembodiments, the subject is of African American (AA) or EuropeanAmerican (EA) descent, i.e., has one or more ancestors who are AA or EA.

In one embodiment, a subject to be evaluated by a method describedherein is a subject having one or more risk factors associated with SZ,SPD or SD. For example, the subject may have a relative afflicted withSZ, e.g., one or more of a grandparent, parent, uncle or aunt, sibling,or child who has or had SZ, SPD or SD; the subject may have agenetically based phenotypic trait associated with risk for SZ, SPD orSD (e.g., eye tracking dysfunction); deficits in working (short-term)memory; and/or mixed-handedness (the use of different hands fordifferent tasks), particularly in females.

In some embodiments, the subject is a child, fetus, or embryo, and oneof the subject's relatives, e.g., a parent or sibling, of the child,fetus, or embryo has SZ, SPD or SD. In this case, the presence in thechild, fetus, or embryo of a haplotype described herein that is sharedwith the affected parent, but not with the non-affected parent,indicates that the child, fetus, or embryo has an increased risk ofdeveloping SPD, SD, or SZ. In some embodiments, the subject has no overtor clinical signs of SZ, SPD, or SD.

In some embodiments, obtaining a test haplotype includes obtaining asample comprising DNA from the subject; and determining the identity,presence or absence of at least one test marker that is listed in TableA, or is within 1 LDU (in the particular population) of a marker listedin Table A, in the DNA. The sample can be obtained, e.g., from thesubject by a health care provider, or provided by the subject withoutthe assistance of a health care provider.

In some embodiments, obtaining a test haplotype includes reviewing asubject's medical history, wherein the medical history includesinformation regarding the presence or absence of at least one testmarker that is listed in Table A, or is within 1 LDU of a marker listedin Table A, in the subject.

In some embodiments, the methods described herein include obtaining areference haplotype including a reference marker that corresponds to atest marker, and comparing the test haplotype to the referencehaplotype. A reference marker that “corresponds to” a test marker is thesame marker. For example, if the test haplotype includes rs313548, thenthe reference haplotype should also include rs313548 for comparisonpurposes; or if the test haplotype includes rs10016449, then thereference haplotype should also include rs10016449 for comparisonpurposes; or if the test haplotype includes rs1548977, then thereference haplotype should also include rs1548977 for comparisonpurposes; or if the test haplotype includes rs9616816, then thereference haplotype should also include rs9616816 for comparisonpurposes.

The sharing of a haplotype (e.g., of some or all of the markers) betweenthe test haplotype and a reference haplotype is indicative of whetherthere is an increased likelihood that the subject will develop SZ, SPD,or SD.

In some embodiments, the methods include administering a treatment to asubject identified as being at increased risk for developing SZ, SPD, orSD, e.g., a pharmacological or psychosocial treatment as describedherein. In some embodiments, the subject has no overt or clinical signsof SZ, SPD, or SD, and the treatment is administrated before any suchsigns appear.

Information obtained using a method described herein can be used, e.g.,to select a subject population for a clinical trial, to stratify asubject population in a clinical trial, and/or to stratify subjects thatrespond to a treatment from those who do not respond to a treatment, orsubjects that have negative side effects from those who do not.

In another aspect, the invention provides methods for selecting asubject for inclusion in a clinical trial, e.g., a trial of a treatmentfor SZ, SPD, or SD. The methods include obtaining a haplotype for thesubject including at least one marker that is listed in Table A, or iswithin 1 linkage disequilibrium unit (1 LDU) of a marker listed in TableA; determining whether the haplotype is associated with an increasedrisk of developing schizophrenia (SZ), schizotypal personality disorder(SPD), or schizoaffective disorder (SD); and including the subject inthe trial if the haplotype indicates that the subject has an increasedrisk of developing SZ, SPD, or SD.

In another aspect, the invention provides methods for selecting asubject for administration of a treatment for schizophrenia (SZ),schizotypal personality disorder (SPD), or schizoaffective disorder(SD). The methods include obtaining a haplotype for the subject, whereinthe haplotype comprises at least one marker that is listed in Table A,or is within 1 linkage disequilibrium unit (1 LDU) of a marker listed inTable A; determining whether the haplotype is associated with anincreased risk of developing SZ, SPD, or SD; and administering thetreatment to the subject if the haplotype indicates that the subject hasan increased risk of developing SZ, SPD, or SD.

In another aspect, the invention provides methods for selecting atreatment for administration to a subject. The methods include obtaininga haplotype for the subject, wherein the haplotype comprises at leastone marker that is listed in Table A, or is within 1 linkagedisequilibrium unit (1 LDU) of a marker listed in Table A; determiningwhether the haplotype is associated with an increased risk of developingschizophrenia (SZ), schizotypal personality disorder (SPD), orschizoaffective disorder (SD); and administering the treatment for SZ,SPD, or SD to the subject if the haplotype indicates that the subjecthas an increased risk of developing SZ, SPD, or SD.

In another aspect, the invention provides methods for evaluating theeffect of a haplotype on the outcome of a treatment for schizophrenia(SZ), schizotypal personality disorder (SPD), or schizoaffectivedisorder (SD). The methods include obtaining information regardingoutcome of the treatment, wherein the information comprises a parameterrelating to the treatment of each subject in a population of subjects;obtaining haplotypes for each subject in the population, wherein thehaplotype comprises at least one marker that is listed in Table A, or iswithin 1 linkage disequilibrium unit (1 LDU) of a marker listed in TableA; and correlating the information regarding outcome with thehaplotypes; thereby evaluating the effect of the haplotype on theoutcome of the treatment.

In some embodiments, the method includes selecting a treatment foradministration to a subject who has a selected haplotype, based on theeffect of the haplotype on the outcome of the treatment.

In some embodiments, the information regarding outcome of the treatmentis from a completed clinical trial, and the analysis is retrospective.

In another aspect, the invention features methods of predicting asubject's risk of developing SZ, SPD, or SD. The methods includeobtaining a reference haplotype. In some embodiments, the referencehaplotype is from at least one of the following relatives of thesubject: (i) a parent who has SZ, SPD, or SD; (ii) a sibling who has SZ,SPD, or SD, and an unaffected parent; or (iii) a second degree relative(e.g., aunt, uncle, or grandparent) who has SZ, SPD, or SD, and anunaffected parent; obtaining a test haplotype from the subject in thesame region; and comparing the test haplotype to a reference haplotype.The sharing of a haplotype in this region between the test haplotype anda reference haplotype from a relative having the disorder is anindication of an increased likelihood that the subject will develop SZ,SPD, or SD. In some embodiments, the reference haplotype is from anunaffected individual, and sharing of a haplotype indicates that thereis no increased likelihood that the subject will develop SZ, SD, or SD.

In a further aspect, the invention features methods for detecting thepresence of a haplotype associated with susceptibility to SZ, SPD, or SDin a subject, by analyzing a sample of DNA from the subject.

Additionally, the invention features methods of predicting a testsubject's risk of developing SZ, SPD, or SD. The methods includeobtaining a reference haplotype of a reference subject, wherein thereference subject has SZ, SPD, or SD; determining a test haplotype ofthe test subject in the same region; and comparing the test haplotype tothe reference haplotype, wherein the sharing of a haplotype in thisregion between the test subject and the reference subject is anindication of an increased likelihood that the test subject will developSZ, SPD, or SD. In some embodiments, the method further includescomparing the subject's haplotype to a reference subject who does nothave SZ, SPD, or SD.

Further, the invention features methods for predicting a test subject'srisk of developing SZ. The methods include obtaining a referencehaplotype of a reference subject in a region described herein, whereinthe reference subject has SZ; obtaining a test haplotype of the testsubject in the same region; and comparing the test haplotype to thereference haplotype. The sharing of a haplotype in this region betweenthe test subject and the reference subject is an indication of anincreased likelihood that the test subject will develop SZ. In someembodiments, the method also includes comparing the test subject'shaplotype to a reference subject who does not have SZ.

In another aspect, the invention features methods for predicting asubject's risk of developing SZ, SPD, or SD. The methods includeobtaining genomic DNA (gDNA) from the subject; and determining theabsence or presence of a haplotype associated with SZ as describedherein. The presence of a haplotype associated with SZ, SPD, or SDindicates that the subject has an increased risk of developing SZ, SD orSPD.

Also provided herein are kits for use in detection of haplotypesassociated with SZ, SD or SPD, including at least one nucleic acid probethat hybridizes to a sequence that includes a polymorphism describedherein, or can be used to amplify a sequence that includes apolymorphism described herein.

Also provided are arrays that include a substrate having a plurality ofaddressable areas, wherein one or more of the addressable areas includesone or more probes that can be used to detect a polymorphism describedherein.

In another aspect, the invention provides methods for providinginformation regarding a subject's risk of developing schizophrenia (SZ),schizotypal personality disorder (SPD), or schizoaffective disorder(SD). The methods include obtaining a sample from the subject at a firstsite; transferring the sample to a second site for analysis, wherein theanalysis provides data regarding the identity, presence or absence of atleast one test marker that is listed in Table A, or is within 1 LDU of amarker listed in Table A; and transferring the data to one or more of ahealth care provider, the subject, or a healthcare payer. In someembodiments, the first site is a health care provider's place ofbusiness, or is not a health care provider's place of business, e.g.,the subject's home.

In some embodiments, the data is transferred to a healthcare payer andused to decide whether to reimburse a health care provider.

DEFINITIONS

As used herein, a “haplotype” is one or a set of signature geneticchanges (polymorphisms) that are normally grouped closely together onthe DNA strand, and are usually inherited as a group; the polymorphismsare also referred to herein as “markers.” A “haplotype” as used hereinis information regarding the presence or absence of one or more geneticmarkers in a subject. A haplotype can consist of a variety of geneticmarkers, including indels (insertions or deletions of the DNA atparticular locations on the chromosome); single nucleotide polymorphisms(SNPs) in which a particular nucleotide is changed; microsatellites; andminisatellites.

Microsatellites (sometimes referred to as a variable number of tandemrepeats or VNTRs) are short segments of DNA that have a repeatedsequence, usually about 2 to 5 nucleotides long (e.g., CACACA), thattend to occur in non-coding DNA. Changes in the microsatellitessometimes occur during the genetic recombination of sexual reproduction,increasing or decreasing the number of repeats found at an allele,changing the length of the allele. Microsatellite markers are stable,polymorphic, easily analyzed and occur regularly throughout the genome,making them especially suitable for genetic analysis.

“Linkage disequilibrium” refers to when the observed frequencies ofhaplotypes in a population does not agree with haplotype frequenciespredicted by multiplying together the frequency of individual geneticmarkers in each haplotype.

The term “chromosome” as used herein refers to a gene carrier of a cellthat is derived from chromatin and comprises DNA and protein components(e.g., histones). The conventional internationally recognized individualhuman genome chromosome numbering identification system is employedherein. The size of an individual chromosome can vary from one type toanother with a given multi-chromosomal genome and from one genome toanother. In the case of the human genome, the entire DNA mass of a givenchromosome is usually greater than about 100,000,000 base pairs. Forexample, the size of the entire human genome is about 3×10⁹ base pairs.Chromosome 22 contains about 5.3×10⁷ base pairs (see, e.g., Yunis,Science 191:1268-1270 (1976), and Kavenoff et al., Cold Spring HarborSymposia on Quantitative Biology 38:1-8 (1973)).

The term “gene” refers to a DNA sequence in a chromosome that codes fora product (either RNA or its translation product, a polypeptide). A genecontains a coding region and includes regions preceding and followingthe coding region (termed respectively “leader” and “trailer”). Thecoding region is comprised of a plurality of coding segments (“exons”)and intervening sequences (“introns”) between individual codingsegments.

The term “probe” refers to an oligonucleotide. A probe can be singlestranded at the time of hybridization to a target. As used herein,probes include primers, i.e., oligonucleotides that can be used to primea reaction, e.g., a PCR reaction.

The term “label” or “label containing moiety” refers in a moiety capableof detection, such as a radioactive isotope or group containing same,and nonisotopic labels, such as enzymes, biotin, avidin, streptavidin,digoxygenin, luminescent agents, dyes, haptens, and the like.Luminescent agents, depending upon the source of exciting energy, can beclassified as radioluminescent, chemiluminescent, bioluminescent, andphotoluminescent (including fluorescent and phosphorescent). A probedescribed herein can be bound, e.g., chemically bound tolabel-containing moieties or can be suitable to be so bound. The probecan be directly or indirectly labeled.

The term “direct label probe” (or “directly labeled probe”) refers to anucleic acid probe whose label after hybrid formation with a target isdetectable without further reactive processing of hybrid. The term“indirect label probe” (or “indirectly labeled probe”) refers to anucleic acid probe whose label after hybrid formation with a target isfurther reacted in subsequent processing with one or more reagents toassociate therewith one or more moieties that finally result in adetectable entity.

The terms “target,” “DNA target,” or “DNA target region” refers to anucleotide sequence that occurs at a specific chromosomal location. Eachsuch sequence or portion is preferably at least partially, singlestranded (e.g., denatured) at the time of hybridization. When the targetnucleotide sequences are located only in a single region or fraction ofa given chromosome, the term “target region” is sometimes used. Targetsfor hybridization can be derived from specimens which include, but arenot limited to, chromosomes or regions of chromosomes in normal,diseased or malignant human cells, either interphase or at any state ofmeiosis or mitosis, and either extracted or derived from living orpostmortem tissues, organs or fluids; germinal cells including sperm andegg cells, or cells from zygotes, fetuses, or embryos, or chorionic oramniotic cells, or cells from any other germinating body; cells grown invitro, from either long-term or short-term culture, and either normal,immortalized or transformed; inter- or intraspecific hybrids ofdifferent types of cells or differentiation states of these cells;individual chromosomes or portions of chromosomes, or translocated,deleted or other damaged chromosomes, isolated by any of a number ofmeans known to those with skill in the art, including libraries of suchchromosomes cloned and propagated in prokaryotic or other cloningvectors, or amplified in vitro by means well known to those with skill;or any forensic material, including but not limited to blood, or othersamples.

The term “hybrid” refers to the product of a hybridization procedurebetween a probe and a target.

The term “hybridizing conditions” has general reference to thecombinations of conditions that are employable in a given hybridizationprocedure to produce hybrids, such conditions typically involvingcontrolled temperature, liquid phase, and contact between a probe (orprobe composition) and a target. Conveniently and preferably, at leastone denaturation step precedes a step wherein a probe or probecomposition is contacted with a target. Guidance for performinghybridization reactions can be found in Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y. (2003),6.3.1-6.3.6. Aqueous and nonaqueous methods are described in thatreference and either can be used. Hybridization conditions referred toherein are a 50% formamide, 2×SSC wash for 10 minutes at 45° C. followedby a 2×SSC wash for 10 minutes at 37° C.

Calculations of “identity” between two sequences can be performed asfollows. The sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in one or both of a first and a secondnucleic acid sequence for optimal alignment and non-identical sequencescan be disregarded for comparison purposes). The length of a sequencealigned for comparison purposes is at least 30%, e.g., at least 40%,50%, 60%, 70%, 80%, 90% or 100%, of the length of the referencesequence. The nucleotides at corresponding nucleotide positions are thencompared. When a position in the first sequence is occupied by the samenucleotide as the corresponding position in the second sequence, thenthe molecules are identical at that position. The percent identitybetween the two sequences is a function of the number of identicalpositions shared by the sequences, taking into account the number ofgaps, and the length of each gap, which need to be introduced foroptimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In some embodiments, the percent identity between twonucleotide sequences is determined using the GAP program in the GCGsoftware package, using a Blossum 62 scoring matrix with a gap penaltyof 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

As used herein, the term “substantially identical” is used to refer to afirst nucleotide sequence that contains a sufficient number of identicalnucleotides to a second nucleotide sequence such that the first andsecond nucleotide sequences have similar activities. Nucleotidesequences that are substantially identical are at least 80%, e.g., 85%,90%, 95%, 97% or more, identical.

The term “nonspecific binding DNA” refers to DNA which is complementaryto DNA segments of a probe, which DNA occurs in at least one otherposition in a genome, outside of a selected chromosomal target regionwithin that genome. An example of nonspecific binding DNA comprises aclass of DNA repeated segments whose members commonly occur in more thanone chromosome or chromosome region. Such common repetitive segmentstend to hybridize to a greater extent than other DNA segments that arepresent in probe composition.

As used herein, the term “stratification” refers to the creation of adistinction between subjects on the basis of a characteristic orcharacteristics of the subjects. Generally, in the context of clinicaltrials, the distinction is used to distinguish responses or effects indifferent sets of patients distinguished according to the stratificationparameters. In some embodiments, stratification includes distinction ofsubject groups based on the presence or absence of particular markers orhaplotypes described herein. The stratification can be performed, e.g.,in the course of analysis, or can be used in creation of distinct groupsor in other ways.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a line graph showing the results of QTL linkage analysis forMMPI-2 schizophrenia scale for chromosome 4. Abscissa shows linkaget-values and ordinate shows position along the chromosome in cM. Themajor peak exceeds the t-value of 4.09 (P=2.2×10⁻⁵), the proposedcriterion for “significant linkage” for complex genetic traits (Kruglyakand Lander, (1995) Am. J. Hum. Genet. 57: 439-454).

FIG. 2 is a line graph showing the results of QTL linkage analysis forMMPI-2 schizophrenia scale for chromosome 22. Abscissa shows linkaget-values and ordinate shows position along the chromosome in cM. Boththe peak at 52 cM and that at 63 cM exceed the t-value of 3.19(P=7×10⁻⁴), proposed criterion for “suggestive” linkage for complexgenetic traits (Kruglyak and Lander, (1995) Am. J. Hum. Genet. 57:439-454).

DETAILED DESCRIPTION

The methods described herein are based, at least in part, on thediscovery of haplotypes and markers that are associated with increasedrisk of having or developing schizophrenia (SZ), schizotypal personalitydisorder (SPD) or schizoaffective disorder (SD). As described herein,analysis provided evidence of association of the disclosed SNPs andhaplotypes with these disorders.

Methods of Diagnoses and Evaluation of Risk

Described herein are a variety of methods for the diagnosis ofsusceptibility to SZ, SPD or SD. “Susceptibility” does not necessarilymean that the subject will develop SZ, SPD or SD, but rather that thesubject is, in a statistical sense, more likely to develop SZ than anaverage member of the population, i.e., has an increased risk ofdeveloping SZ, SPD, or SD. As used herein, susceptibility to SZ existsif the subject has a haplotype associated with an increased risk of SZ,SPD, or SD as described herein. Ascertaining whether the subject hassuch a haplotype is included in the concept of diagnosing susceptibilityto SZ, SPD or SD as used herein. Such determination is useful, forexample, for purposes of diagnosis, treatment selection, and geneticcounseling. Thus, the methods described herein can include obtaining ahaplotype associated with an increased risk of SZ, SPD, or SD asdescribed herein for the subject.

As used herein, “obtaining a haplotype” includes obtaining informationregarding the identity, presence or absence of one or more geneticmarkers in a subject. Obtaining a haplotype can, but need not, includeobtaining a sample comprising DNA from a subject, and/or assessing theidentity, presence or absence of one or more genetic markers in thesample. The individual or organization who obtains the haplotype neednot actually carry out the physical analysis of a sample from a subject;the haplotype can include information obtained by analysis of the sampleby a third party. Thus the methods can include steps that occur at morethan one site. For example, a sample can be obtained from a subject at afirst site, such as at a health care provider, or at the subject's homein the case of a self-testing kit. The sample can be analyzed at thesame or a second site, e.g., at a laboratory or other testing facility.

Obtaining a haplotype can also include or consist of reviewing asubject's medical history, where the medical history includesinformation regarding the identity, presence or absence of one or moregenetic markers in the subject, e.g., results of a genetic test.

In some embodiments, to detect the presence of a haplotype describedherein, a biological sample that includes nucleated cells (such asblood, a cheek swab or mouthwash) is prepared and analyzed for thepresence or absence of preselected markers. Such diagnoses may beperformed by diagnostic laboratories, or, alternatively, diagnostic kitscan be manufactured and sold to health care providers or to privateindividuals for self-diagnosis. Diagnostic or prognostic tests can beperformed as described herein or using well known techniques, such asdescribed in U.S. Pat. No. 5,800,998.

Results of these tests, and optionally interpretive information, can bereturned to the subject, the health care provider or to a third partypayor. The results can be used in a number of ways. The information canbe, e.g., communicated to the tested subject, e.g., with a prognosis andoptionally interpretive materials that help the subject understand thetest results and prognosis. The information can be used, e.g., by ahealth care provider, to determine whether to administer a specificdrug, or whether a subject should be assigned to a specific category,e.g., a category associated with a specific disease endophenotype, orwith drug response or non-response. The information can be used, e.g.,by a third party payor such as a healthcare payer (e.g., insurancecompany or HMO) or other agency, to determine whether or not toreimburse a health care provider for services to the subject, or whetherto approve the provision of services to the subject. For example, thehealthcare payer may decide to reimburse a health care provider fortreatments for SZ, SPD or SD if the subject has an increased risk ofdeveloping SZ, SPD or SD. As another example, a drug or treatment may beindicated for individuals with a certain haplotype, and the insurancecompany would only reimburse the health care provider (or the insuredindividual) for prescription or purchase of the drug if the insuredindividual has that haplotype. The presence or absence of the haplotypein a patient may be ascertained by using any of the methods describedherein.

Information gleaned from the methods described herein can also be usedto select or stratify subjects for a clinical trial. For example, thepresence of a selected haplotype described herein can be used to selecta subject for a trial. The information can optionally be correlated withclinical information about the subject, e.g., diagnostic orendophenotypic information.

Haplotypes Associated with SZ, SPD and SD

As described herein, haplotypes associated with SZ, SPD or SD includemarkers eg. in 4p15.31 (KCNIP4), as exemplified by the transmissiondisequilibrium results shown in tables 4 and 5; eg. in 4p15.2 (PI4K2B),as exemplified by the transmission disequilibrium results shown intables 6 and 7; eg. in 22q13.31 (CERK) as exemplified by thetransmission disequilibrium results shown in tables 9 and 10; eg. in22q13.33 (SHANK3) as exemplified by the transmission disequilibriumresults shown in tables 11 and 12.

As one example, haplotypes associated with a broader disorder definitionincluding SZ, SPD and SD include one or more markers on chromosomes 4por 22q that are within 1 linkage disequilibrium unit (1 LDU) of a markerlisted in Tables 4, 5, 6, 7, 9, 10, 11 or 12. In some embodiments, thehaplotype includes one or more of the markers listed in Tables 4, 5, 6,7, 9, 10, 11 or 12. Haplotypes associated with a broader disorderdefinition of SZ can include one or more markers that are within 1 LDUof a marker listed in Tables 4, 5, 6, 7, 9, 10, 11 or 12. In someembodiments, the markers are in a region of 4p15.2 that is between andincluding SNPs rs313548 and rs313567 at the PI4K2B locus. In someembodiments, the markers are in a region of 4p15.31 between rs6447982and rs1364836 at the KCNIP4 locus. In some embodiments, the markers arein a region of 22q13.31 between rs801720 and rs710123 at the CERK locus.In some embodiments, the markers are in a region of 22q13.33 betweenrs713692 and rs756638 at the SHANK3 locus.

As one example, haplotypes associated with a narrow disease definitionof SZ include one or more markers on chromosomes 4p or 22q that arewithin 1 linkage disequilibrium unit (1 LDU) of a marker listed inTables 4, 5, 6, 7, 9, 10, 11 or 12. Haplotypes associated with anarrower disorder definition of SZ can include one or more markers thatare within 1 LDU of a marker listed in Tables 4, 5, 6, 7, 9, 10, 11 or12. In some embodiments, the markers are in a region of 4p15.2 that isbetween and including SNPs rs313548 and rs313567 at the PI4K2B locus. Insome embodiments, the markers are in a region of 4p15.31 betweenrs6447982 and rs1364836 at the KCNIP4 locus. In some embodiments, themarkers are in a region of 22q13.31 between rs801720 and rs710123 at theCERK locus. In some embodiments, the markers are in a region of 22q13.33between rs713692 and rs756638 at the SHANK3 locus.

In some embodiments, the gene is KCNIP4, and thers6447982(A)/rs10016449(T) haplotype is associated with disease. In someembodiments, the gene is PI4K2B, and the rs313548(C) allele isassociated with disease. In some embodiments, the gene is CERK, and thers135667(G)/rs1548977(A) haplotype is associated with disease. In someembodiments, the gene is SHANK3, and the rs9616816(A)-rs6010063(A)haplotype is associated with disease.

In some embodiments, the methods include determining the presence of ahaplotype that includes one or more polymorphisms near D22S526 and/orthe polymorphisms in the Sult4a1 gene listed in Table 4, and/orpolymorphisms within 1 LDU of these markers, e.g., as described in U.S.Pat. Pub. No. 2006-0177851, incorporated herein in its entirety.

SH3 and Multiple Ankyrin Repeat Domains 3 (SHANK3)

SH3 and multiple ankyrin repeat domains 3 (SHANK3, also known as PSAP2;PROSAP2; SPANK-2; and KIAA1650) is a synaptic scaffolding protein thatregulates the structural organization of dendritic spines and is abinding partner of proteins known as neuroligins. The human mRNA andprotein sequences of SHANK3 are available in GenBank atNM_(—)001080420.1 and NP_(—)001073889.1, respectively. The genomicsequence can be found at NC_(—)000022.9 in Genome Build 36.2(nucleotides 49459936-49518507 of chromosome 22), with an alternateassembly (based on Celera assembly) at AC_(—)000065.1. For additionalinformation, see also UniGene entry no. Hs.149035 and GeneID: 85358 inthe Entrez Gene database. Previously, a role of SHANK3 in autismspectrum disorders has been speculated. See, e.g., Durand et al., Nat.Genet. 39(1):25-7 (2007) [Epub 2006 Dec. 17]

Ceramide Kinase (CERK)

Ceramide kinase (CERK, also known as LK4; hCERK; FLJ21430; FLJ23239;KIAA1646; MGC131878; dA59H18.2; dA59H18.3; and DKFZp434E0211). The humanCERK has two isoforms, A and B. The mRNA for isoform A is available inGenBank at NM_(—)022766.4, and the protein is at NP_(—)073603.2. IsoformB is NM_(—)182661.1 (mRNA) and NP_(—)872602.1 (protein) that isdevelopmentally regulated and shows subcellular location-dependentactivity. The genomic sequence is NC_(—)000022.9 assembly in Build 36.2,and is at nucleotides 45512816-45458971 of chromosome 22. For additionalinformation see GeneID: 64781 and UniGene: Hs.200668.

Phosphatidylinositol 4-Kinase Type 2 Beta (PI4K2B)

Phosphatidylinositol 4-Kinase Type 2 Beta (PI4K2B, also known as PIK42B;PI4KIIB; and FLJ11105) is an enzyme that can phosphorylate and removephosphatidylinositol-5-phosphate and may be involved in the response tocellular stress. The human mRNA and protein sequences are in Genbank atNM_(—)018323.2 and NP_(—)060793.1, respectively. The genomic Referenceassembly is NC_(—)000004.10 in build 36.2, nucleotides 24844773-24889808of chromosome 4. See GeneID: 55300 and UniGene Hs.638037 for additionalinformation.

Kv Channel Interacting Protein 4 (KCNIP4)

Kv channel interacting protein 4 (KCNIP4, also known as CALP; KCHIP4;and MGC44947) encodes a member of the family of voltage-gated potassium(Kv) channel-interacting proteins (KCNIPs). Members of the KCNIP familyare small calcium binding proteins that are subunit components of nativeKv4 channel complexes, and may regulate A-type currents, and thusneuronal excitability, in response to changes in intracellular calciumlevels. KCNIP4 also interacts with presenilin. At least 5 alternativelyspliced transcript variants encoding distinct isoforms exist for thisgene, as follows:

mRNA GenBank Protein GenBank Acc. No. Acc. No Name NM_001035003.1NP_001030175.1 Kv channel interacting protein 4 isoform 5 NM_001035004.1NP_001030176.1 Kv channel interacting protein 4 isoform 6 NM_025221.5NP_079497.2 Kv channel interacting protein 4 isoform 1 NM_147182.3NP_671711.1 Kv channel interacting protein 4 isoform 3 NM_147181.3NP_671710.1 Kv channel interacting protein 4 isoform 2 NM_147183.3NP_671712.1 Kv channel interacting protein 4 isoform 4

The Reference assembly of the genomic sequence is NC_(—)000004.10,nucleotides 21155377-20339337 of build 36.2 of chromosome 4 are thecomplement. An alternate assembly (based on the Celera assembly) is atAC_(—)000047.1, nucleotides 21996734-21186744 (complement).

Linkage Disequilibrium Analysis

Linkage disequilibrium (LD) is a measure of the degree of associationbetween alleles in a population. One of skill in the art will appreciatethat haplotypes involving markers within 1 Linkage Disequilibrium Unit(LDU) of the polymorphisms described herein can also be used in asimilar manner to those described herein. LDUs share an inverserelationship with LD so that regions with high LD (such as haplotypeblocks) have few LDUs and low recombination, whilst regions with manyLDUs have low LD and high recombination. Methods of calculating LDUs areknown in the art (see, e.g., Morton et al., Proc Natl Acad Sci USA98(9):5217-21 (2001); Tapper et al., Proc Natl Acad Sci USA102(33):11835-11839 (2005); Maniatis et al., Proc Natl Acad Sci USA99:2228-2233 (2002)).

Thus, in some embodiments, the methods include analysis of polymorphismsthat are within 1 LDU of a polymorphism described herein. Methods areknown in the art for identifying such polymorphisms; for example, theInternational HapMap Project provides a public database that can beused, see hapmap.org, as well as The International HapMap Consortium,Nature 426:789-796 (2003), and The International HapMap Consortium,Nature 437:1299-1320 (2005). Generally, it will be desirable to use aHapMap constructed using data from individuals who share ethnicity withthe subject, e.g., a HapMap for African Americans would ideally be usedto identify markers within 1 LDU of a marker described herein for use ingenotyping a subject of African American descent.

Exemplary polymorphisms that are within 1 LDU of some of the markersdescribed herein are included in the Examples, e.g., Example 6.

Alternatively, methods described herein can include analysis ofpolymorphisms that are within a value defined by Lewontin's D′ (linkagedisequilibrium parameter, see Lewontin, Genetics 49:49-67 (1964)) of apolymorphism described herein. Results can be obtained, e.g., from online public resources such as HapMap.org. The simple linkagedisequilibrium parameter (D) reflects the degree to which alleles at twoloci (for example two SNPs) occur together more often (positive values)or less often (negative values) than expected in a population asdetermined by the products of their respective allele frequencies. Forany two loci, D can vary in value from −0.25 to +0.25. However, themagnitude of D (Dmax) varies as function of allele frequencies. Tocontrol for this, Lewontin introduced the D′ parameter, which is D/Dmaxand varies in value from −1 (alleles never observed together) to +1(alleles always observed together). Typically, the absolute value of D′(i.e., |D′|) is reported in online databases, because it followsmathematically that positive association for one set of alleles at twoloci corresponds to a negative association of equal magnitude for thereciprocal set. This disequilibrium parameter varies from 0 (noassociation of alleles at the two loci) to 1 (maximal possibleassociation of alleles at the two loci).

Thus, in some embodiments, the methods include analysis of polymorphismsthat are within D′>0.75, or D′=1, for pairwise comparisons, of apolymorphism described herein.

Identification of Additional Markers for Use in the Methods DescribedHerein

In general, genetic markers can be identified using any of a number ofmethods well known in the art. For example, numerous polymorphisms inthe regions described herein are known to exist and are available inpublic databases, which can be searched using methods and algorithmsknown in the art. Alternately, polymorphisms can be identified bysequencing either genomic DNA or cDNA in the region in which it isdesired to find a polymorphism. According to one approach, primers aredesigned to amplify such a region, and DNA from a subject is obtainedand amplified. The DNA is sequenced, and the sequence (referred to as a“subject sequence” or “test sequence”) is compared with a referencesequence, which can represent the “normal” or “wild type” sequence, orthe “affected” sequence. In some embodiments, a reference sequence canbe from, for example, the human draft genome sequence, publiclyavailable in various databases, or a sequence deposited in a databasesuch as GenBank. In some embodiments, the reference sequence is acomposite of ethnically diverse individuals.

In general, if sequencing reveals a difference between the sequencedregion and the reference sequence, a polymorphism has been identified.The fact that a difference in nucleotide sequence is identified at aparticular site that determines that a polymorphism exists at that site.In most instances, particularly in the case of SNPs, only twopolymorphic variants will exist at any location. However, in the case ofSNPs, up to four variants may exist since there are four naturallyoccurring nucleotides in DNA. Other polymorphisms, such as insertionsand deletions, may have more than four alleles.

Other Genetic Markers of Schizophrenia

The methods described herein can also include determining the presenceor absence of other markers known or suspected to be associated with SZ,or with SZ, SD or SPD, e.g., markers outside of a region identifiedherein, see, e.g., Harrison and Owen, Lancet, 361(9355):417-419 (2003),including, for example, markers on chromosome 22 and other chromosomes,e.g., in the region of 22q12.3 (e.g., near D22S283), 22q11.2, 22q11.2,22q11-q13, 1q42.1, 1q42.1, 4p, 18p, 15q15, 14q32.3, 13q34, 13q32, 12q24,11q14-q21, 1q21-q22, 10p15-p13 (e.g., near D105189), 10q22.3, 8p12-21,6q13-q26, 6p22.3, 6p23, 5q11.2-q13.3, and/or 3p25. In some embodiments,the methods include determining the presence or absence of one or moreother markers that are or may be associated with SZ, or with SZ, SD orSPD, e.g., in one or more genes, e.g., ACE (Illi et al., EurNeuropsychopharmacol 13:147-151 (2003)); ADRA1A (Clark et al., BiolPsychiatry. 58(6):435-9 (2005)); ADH1B (Xu et al., Mol. Psychiatry.9(5):510-21 (2004); Vawter et al., Hum Genet. 119(5):558-70 (2006));AHI1 (Eur J Hum Genet. 14(10):1111-9 (2006)); AKT1 (Emamian et al.,Nature Genet. 36:131-137 (2004)); ALDH3B1 (Sun et al. Sci. China C.Life. Sci. 48(3):263-9 (2005)); ALK (Kunagi et al., J Neural Transm.113(10):1569-73 (2006)); APC (Cui et al., Mol Psychiatry (7):669-77(2005)); APOE (Liu et al., Schizophr Res 62: 225-230 (2003)); ARSA(Marcao et al., Mol Genet Metab. 79(4):305-7 (2003); ARVCF (Chen et al.,Schizophr Res. 72(2-3):275-7 (2005)); ATXN1 (Pujana et al Hum Genet99:772-775 (1997); Joo et al., Psychiatr Genet 9:7-11 (1999); Fallin etal., Am J Hum Genet 77:918-936 (2005)); BDNF (Neves-Pereira et al.,Molec. Psychiat. 10:208-212 (2005)); BRD1 (Severinsen et al., Mol.Psychiatry. 11(12):1126-38 (2006)); BZRP (Kurumaji et al., J NeuralTransm. 107(4):491-500 (2000)); DAO (Owen et al., Trends Genet.21(9):518-25 (2005)); DAOA (Owen et al., 2005, supra); CAPON(Brzustowicz et al., Am J Hum Genet. 74(5):1057-63 (2004)); CCKAR (Zhanget al., Mol Psychiatry 5:239-240 (2000); Sanjuan et al., Eur Psychiatry19:349-353 (2004)); CHGB (Kitao et al., Psychiatr Genet 10:139-143(2000); Zhang et al., Neurosci Lett 323:229-233 (2002)); CHI3L1 (Zhao etal., Am J Hum Genet. 80(1):12-8 (2007)); CHRNA2 (Blayeri et al., Europ.J. Hum. Genet. 9: 469-472 (2001)); CHRNA7 (Leonard et al. Arch GenPsychiatry. 2002 59:1085-1096 (2002); De Luca et al. Neuropsychobiology.50:124-127 (2004)); CLDN5 (Sun et al., Eur Psychiatry 19:354-357 (2004);Wei and Hemmings, Prostaglandins Leukot Essent Fatty Acids 73(6)4:41-445(2005)); COMT (Shifman et al., Am. J. Hum. Genet. 71:1296-1302 (2002));CNR1 (Ujike et al., Mol Psychiatry 7:515-518 (2002)); CPLX2 (Lee et al.,Behav Brain Funct. 1:15 (2005)); DGCR8 (Jacquet et al., Hum Mol. Genet.11(19):2243-9 (2002)); DISC1 (Owen et al., 2005, supra; see, e.g., theD1S2709 marker (Ekelend et al., Hum. Molec. Genet. 10:1611-1617 (2001),DDR1 (Roig et al., Mol. Psychiatry. 2007 Apr. 17; [Epub ahead ofprint]); DRD4 (Lung et al., Schizophr Res 57:239-245 (2002)); DDR3(Williams et al., Mol Psychiatry 3:141-149 (1998)); DRD5 (Williams etal., Psychiatr Genet 7:83-85 (1997); Muir et al., Am J Med Genet105:152-158 (2001)); HEP3 haplotype, Hennah et al., Hum. Molec. Genet.12: 3151-3159 (2003), and Leu607Pro, Hodgkinson et al., Am. J. Hum.Genet. 75:862-872 (2004), Erratum: Am. J. Hum. Genet. 76:196 (2005));DISC2 (Millar et al., Ann Med. 36(5):367-78 (2004)); DPYSL2 (Hong etal., Am J Med Genet B Neuropsychiatr Genet. 136(1):8-11 (2005)); DRD1(Coon et al., Am. J. Hum. Genet. 52: 327-334 (1993)); DRD2 (Glatt etal., Am. J. Psychiat. 160:469-476 (2003)); DRD3 (Rybakowski et al.,Molec. Psychiat. 6:718-724 (2001)); DTNBP1 (Owen et al., 2005, supra);EGR3 (Yamada et al., Proc Natl Acad Sci 104(8):2815-20 (2007)); EPSIN4(Am J Hum Genet. 76(5):902-7 (2005)); ErbB; EGF (Futamura et al., Am. J.Hum. Genet. 52: 327-334 (2002)); ENTH (Pimm et al., Am J Hum Genet76:902-907 (2005); Tang et al., Mol Psychiatry 11:395-399 (2006)); ERBB4(Norton et al., Am J Med Genet B Neuropsychiatr Genet 14; 11; 96-101(2005); Silberberg et al., Am J Med Genet B Neuropsychiatr Genet 141B;2; 142-148 (2006)); FEZ1 (Yamada et al., Biol Psychiatry56:683-690(2004)); FOXP2 (Sanjuan et al., Psychiatr Genet. 16(2):67-72(2006)); FXYD6 (Choudhury et al., Am J Hum Genet. 80(4):664-72 (2007));FZD3 (Katsu et al., Neurosci Lett 353:53-56 (2003); Yang et al., BiolPsychiatry 54:1298-1301 (2003); Zhang et al., Am J Med Genet 129B:16-19(2004)); GABRA1, GABRA2, GABRA6, GABRP (Petryshen et al., Mol.Psychiatry. 10(12):1057 (2005)); GABBR1 (Zai et al. EurNeuropsychopharmacol. 15:347-52 (2005); Le-Niculescu et al. Am J MedGenet B Neuropsychiatr Genet. 144:129-58 (2007)); GAD1 (Addington etal., Mol Psychiatry 10:581-588 (2005)); GFRA1 (Semba et al., Brain ResMol Brain Res. 124(1):88-95 (2004)); GCLM (Tosic et al., Am J Hum Genet.79(3):586-92 (2006)); GNB3 (Kunugi et al., J. Neural Transm.109(2):213-8 (2002)); GPR78 (Underwood et al., Mol. Psychiatry.11(4):384-94 (2006)); GRIA1 (Magri et al., Am J Med Genet BNeuropsychiatr Genet 141(3):287-93 (2006)); GNPAT (Lin et al., BiolPsychiatry. 60(6):554-62 (2006)); GRID1 (Fallin et al., Am J Hum Genet77:918-936 (2005)); GRIK1 (Shibata et al., Psychiatr Genet. 11(3):139-44(2001)); GRIK2 (Shibata et al., Psychiatry Res. 113(1-2):59-67 (2002));GRIK3 (Shibata et al., Psychiatry Res. 30: 141(1): 39-51 (2006)); GRIK4(Pikard et al., Mol Psychiatry 11(9):847-57 (2006)); GRIN1 (Qin et al.,Eur J Hum Genet. 13(7):807-14 (2005)); GRIN2A, GRIN2B (Abdolmaleky etal., Am J Pharmacogenomics. 5(3):149-60 (2005)); GRIN2D (Makino et al.,Psychiatr Genet. 15(3):215-21 (2005)); GRM3 (Egan et al., Proc Natl AcadSci USA. 101(34):12604-9 (2004)); GRM4 (Ohtsuki et al., Psychiatr Genet.11(2):79-83 (2001)); GRM5 (Devon et al., Mol. Psychiatry. 6(3):311-4(2001)); GSTM1 (Harada et al., Biochem Biophys Res Commun 281:267-271(2001); Pae et al., Psychiatr Genet 14:147-150 (2004)); G30/G72 (Schulzeet al., Am J Psychiatry. 162(11):2101-8 (2005)); HTR2A (Baritaki et al.,Eur J Hum Genet. 12(7):535-41 (2004)); HLA-DRB1 (Schwab et al., Am J MedGenet. 114(3):315-20 (2002)); HLA-BRB3 (Yu et al., Zhonghua Liu XingBing Xue Za Zhi. 24(9):815-8 (2003)); HTR5A (Abdolmaleky et al.,Schizophr Res 67:53-62 (2004)); HTR6 (Tsai et al., Neurosci Lett.271(2):135-7 (1999)); IL1B (Katila et al., Mol Psychiatry 4:179-181(1999); Meisenzahal et al., Am J Psychiatry 158:1316-1319 (2001);Zanardini et al., J Psychiatr Res 37:457-462 (2003)); IL1RN (Zanardiniet al., J Psychiatr Res 37:457-462 (2003); Kim et al., Psychiatr Genet14:165-167 (2004); Papiol et al., Neuroimage 27:1002-1006 (2005)); IL10(Chiavetto et al., Biol Psychiatry 51:480-484 (2002); Jun et al.,Psychiatry Clin Neurosci 56:177-180 (2002)); IL2RB (Schwab et al., Am JMed Genet. 60(5):436-43 (1995)); KCNN3 (Ujike et al., Psychiatry Res.101(3):203-7 (2001)); KIF13A (Jamain et al., Genomics. 74(1):36-44(2001)); KIF2A (Li et al., Neurosci Letters 407(2) 151-5 (2006)); KPNA3(Wei and Hemmings, Neurosci Res. 52(4):342-6 (2005)); LGI1 (Fallin etal. A J Hum Genet. 77:918-36 (2005)); MAG (Wan et al., Neurosci Lett.388(3):126-31 (2005)); MAOA (Jonsson et al., Schizophr Res 61:31-37(2003); Wei and Hemmings. Psychiatr Genet 9, 177-181 (1999)); MED12(Sandhu et al., Am J Med Genet B Neuropsychiatr Genet. 123B: 33-38(2003); Spinks et al., Am J Med Genet B Neuropsychiatr Genet. 127B:20-27(2004)); MLC1 (Verma et al., Biol Psychiatry. 58(1):16-22 (2005)); MTHFR(Lewis et al., Am. J. Med. Genet. (Neuropsychiat. Genet.) 135B:2-4(2005)); MTR (Kempisty et al., Psychiatr Genet. 17(3):177-81 (2007));MTHFD1 (Kempisty et al., Psychiatr Genet. 17(3):177-81 (2007)); NCAM1(Sullivan et al., Biol Psychiatry. 61(7):902-10 (2007)); NDE1 (Hennah etal., Hum Mol. Genet. 16(5):453-62 (2006)); NDUFV2 (Waskizuka et al., AmJ Med Genet B Neuropsychiatr Genet. 141(3):301-4 (2006)); NOS1 (Liou etal., Schizophr Res. 65(1):57-9 (2003)); NOTCH4 (Wei and Hemmings,(Letter) Nature Genet. 25:376-377 (2000)); NPAS3 (Kamnasaran et al., JMed Genet 40:325-332 (2003)); NRG1 (Owen et al., 2005, supra); NRG3(Fallin et al. A J Hum Genet. 77:918-36 (2005)); NTNG1 (Fukawasa et al.,J Med Dent Sci 51:121-128 (2004); Aoki-Suzuki et al., Biol Psychiatry57:382-393 (2005)); NTNG2 (Aoki-Suzuki et al., Biol Psychiatry57:382-393 (2005)); NTF3 (Jonsson et al., Acta Psychiatr Scand95:414-419 (1997)); OLIG2 (Georgieva et al., Proc Natl Acad Sci103(33):12469-74 (2006)); PCQAP (Sandhu et al., Psychiatr Genet.14(3):169-72 (2004)); PDE4B (Millar et al., Science 310:1187-1191(2005)); PDLIM5 (Horiuchi et al., Biol Psychiatry 59(5):434-9 (2005));PICK1 (Hong et al., Neuroreport 15:1965-1967 (2004); Fujii et al.,Molecular Psychiatry 11:150-157 (2005)); PIK3C3 (Stopkova et al., BiolPsychiatry 55:981-988 (2004); Duan et al., Neurosci Lett., 379:32-36(2005)); PIK4CA (Saito et al., Am J Med Genet B Neuropsychiatr Genet.116(1):77-83 (2003)); PIP5K2A (Stopkova et al., Psychiatr Genet. 15(3):223-7 (2005)); PLA2G4A, PLA2G4C (Yu et al., Prostaglandins Leukot EssentFatty Acids. 73(5):351-4 (2005)); PLA2G4B (Tao et al., Am J Med Genet BNeuropsychiatr Genet 137:56-58 (2005)); PLXNA2 (Mah et al., MolecularPsychiatry 11:471-478 (2006)); PTGS2 (Wei and Hemmings. ProstaglandinsLeukot Essent Fatty Acids 70:413-415 (2004)); PPP3CC (Gerber et al.,Proc Natl Acad Sci USA. 100(15):8993-8 (2003)); PNOC (Blayeri et al.,2001); PRODH (Chakravarti, Proc. Nat. Acad. Sci. 99:4755-4756 (2002));QKI (Aberg et al., Am J Med Genet B Neuropsychiatr Genet. 2005 Dec. 9;[Epub ahead of print]); RGS4 (Chowdari et al., Hum. Molec. Genet.11:1373-1380 (2002), Erratum: Hum. Molec. Genet. 12:1781 (2003)); RELN(Costa et al., Mol. Interv. 2(1):47-57 (2002)); RTN4 (Novak et al.,Brain Res Mol Brain Res 107:183-189 (2002); Tan et al., Brain Res MolBrain Res 139:212-216 (2005)); SCAT (Culkjovic et al., Am J Med Genet.96(6):884-7 (2000)); SLC15A1 (Maheshwari et al., BMC Genomics. 3(1):30(2002)); SLC18A1 (Bly, Schizophr Res. 78(2-3):337-8 (2005)); SLC18A2(Gutierrez et al. Am J Med Genet B Neuropsychiatr Genet. 144(4):502-7(2007)); SLC6A4 (Fan and Sklar, Mol. Psychiatry. 10(10):928-38, 891(2005)); SNAP29 (Saito et al., Mol Psychiatry 6(2):193-201 (2001);Erratum in: Mol Psychiatry 6(5):605 (2001); SULT4A1 (Brennan andChondra. Am J Med Genet B Neuropsychiatr Genet. 139(1):69-72 (2005));SYNGR1 (Verma et al., Biol Psychiatry. 55(2):196-9 (2004)); SYN2 (Chenet al., Bio. Psychiat. 56:177-181 (2004)); SYN3 (Porton et al. BiolPsychiatry. 55(2):118-25 (2004)); TAAR4 (Duan et al., Am J Hum Genet75:624-638 (2004)); TBP/SCA17 (Chen et al., Schizophr Res. 78(2-3):131-6(2005)); TH (Kurumaji et al., J Neural Transm 108:489-495 (2001); Meloniet al., C R Acad Sci III 318:803-809 (1995)); TNFA (Morar et al., Am JMed Genet B Neuropsychiatr Genet. 144(3):318-24 (2007)); TPH1 (Nolan etal., Psychiatr Genet 10:109-115 (2000); Hong et al., Schizophr Res49:59-63 (2001); Sekizawa et al., Am J Med Genet B Neuropsychiatr Genet128:24-26 (2004)); TPP2 (Fallin et al. A J Hum Genet. 77:918-36 (2005));TPS3 (Park et al., Schizophr Res 67:71-74 (2004); Ni et al., NeurosciLett 388:173-178 (2005)); TRAR4 (Am J Hum Genet. 75(4):624-38 (2004));TRAX (Thomson et al., Mol. Psychiatry. 10(7):657-68, 616 (2005)); UFD1L(De Luca et al., Am J Med Genet. 105(6):529-33 (2001)); UCP2 (Yasuno etal., Am J Med Genet B Neuropsychiatr Genet. 144(2):250-3 (2007)); UCP4(Yasuno et al., : Am J Med Genet B Neuropsychiatr Genet. 144(2):250-3(2007)); UHMK1 (Puri et al., Biol Psychiatry 61(7):873-9 (2007)); XBP1(Chen et al., Biochem Biophys Res Commun 319:866-870 (2004); Kakiuchi etal., Psychiatry Clin Neurosci 58:438-440 (2004)); YWHAH (Toyooka et al.,Am J Med Genet. 88(2):164-7 (1999)); ZDHHC8 (Mukai et al., Nature Genet.36:725-731 (2004)); or ZNF74 (Takase et al., Schizophr Res. 52(3):161-5(2001)). See also, e.g., OMIM entry no. 181500 (SCZD).

In some embodiments, the methods described herein can includedetermining the presence or absence of a haplotype associated with SZ,SPD or SD, as described in U.S. Pat. Pub. No. 2006-0177851, the entirecontents of which are incorporate herein by reference. For example, thehaplotype can include one or more markers in a region of 22q13 that isbetween and including SNPs rs738596 on the proximal end, and rs137853 onthe distal end. For example, the haplotype can include marker D22S526,and/or a polymorphism of Sulfotransferase 4A1 (Sult4a1), e.g., one ormore of rs138060, rs138097, rs138110, or D22s1749e.

Methods of Determining the Presence or Absence of a Haplotype Associatedwith SZ, SPD or SD

The methods described herein include determining the presence or absenceof haplotypes associated with SZ, SPD or SD. In some embodiments, anassociation with SZ is determined by the presence of a shared haplotypebetween the subject and an affected reference individual, e.g., a firstor second-degree relation of the subject, and the absence of thehaplotype in an unaffected reference individual. Thus the methods caninclude obtaining and analyzing a sample from a suitable referenceindividual.

Samples that are suitable for use in the methods described hereincontain genetic material, e.g., genomic DNA (gDNA). Non-limitingexamples of sources of samples include urine, blood, and tissue. Thesample itself will typically consist of nucleated cells (e.g., blood orbuccal cells), tissue, etc., removed from the subject. The subject canbe an adult, child, fetus, or embryo. In some embodiments, the sample isobtained prenatally, either from a fetus or embryo or from the mother(e.g., from fetal or embryonic cells in the maternal circulation).Methods and reagents are known in the art for obtaining, processing, andanalyzing samples. In some embodiments, the sample is obtained with theassistance of a health care provider, e.g., to draw blood. In someembodiments, the sample is obtained without the assistance of a healthcare provider, e.g., where the sample is obtained non-invasively, suchas a sample comprising buccal cells that is obtained using a buccal swabor brush, or a mouthwash sample.

The sample may be further processed before the detecting step. Forexample, DNA in a cell or tissue sample can be separated from othercomponents of the sample. The sample can be concentrated and/or purifiedto isolate DNA. Cells can be harvested from a biological sample usingstandard techniques known in the art. For example, cells can beharvested by centrifuging a cell sample and resuspending the pelletedcells. The cells can be resuspended in a buffered solution such asphosphate-buffered saline (PBS). After centrifuging the cell suspensionto obtain a cell pellet, the cells can be lysed to extract DNA, e.g.,gDNA. See, e.g., Ausubel et al., 2003, supra. All samples obtained froma subject, including those subjected to any sort of further processing,are considered to be obtained from the subject.

The absence or presence of a haplotype associated with SZ, SPD or SD asdescribed herein can be determined using methods known in the art, e.g.,gel electrophoresis, capillary electrophoresis, size exclusionchromatography, sequencing, and/or arrays to detect the presence orabsence of the marker(s) of the haplotype. Amplification of nucleicacids, where desirable, can be accomplished using methods known in theart, e.g., PCR.

Methods of nucleic acid analysis to detect polymorphisms and/orpolymorphic variants include, e.g., microarray analysis. Hybridizationmethods, such as Southern analysis, Northern analysis, or in situhybridizations, can also be used (see Current Protocols in MolecularBiology, Ausubel, F. et al., eds., John Wiley & Sons 2003). To detectmicrodeletions, fluorescence in situ hybridization (FISH) using DNAprobes that are directed to a putatively deleted region in a chromosomecan be used. For example, probes that detect all or a part of amicrosatellite marker can be used to detect microdeletions in the regionthat contains that marker.

Other methods include direct manual sequencing (Church and Gilbert,Proc. Natl. Acad. Sci. USA 81:1991-1995 (1988); Sanger et al., Proc.Natl. Acad. Sci. 74:5463-5467 (1977); Beavis et al. U.S. Pat. No.5,288,644); automated fluorescent sequencing; single-strandedconformation polymorphism assays (SSCP); clamped denaturing gelelectrophoresis (CDGE); two-dimensional gel electrophoresis (2DGE orTDGE); conformational sensitive gel electrophoresis (CSGE); denaturinggradient gel electrophoresis (DGGE) (Sheffield et al., Proc. Natl. Acad.Sci. USA 86:232-236 (1989)), mobility shift analysis (Orita et al.,Proc. Natl. Acad. Sci. USA 86:2766-2770 (1989)), restriction enzymeanalysis (Flavell et al., Cell 15:25 (1978); Geever et al., Proc. Natl.Acad. Sci. USA 78:5081 (1981)); quantitative real-time PCR (Raca et al.,Genet Test 8(4):387-94 (2004)); heteroduplex analysis; chemical mismatchcleavage (CMC) (Cotton et al., Proc. Natl. Acad. Sci. USA 85:4397-4401(1985)); RNase protection assays (Myers et al., Science 230:1242(1985)); use of polypeptides that recognize nucleotide mismatches, e.g.,E. coli mutS protein; allele-specific PCR, for example. See, e.g., U.S.Patent Publication No. 2004/0014095, to Gerber et al., which isincorporated herein by reference in its entirety. In some embodiments,the methods described herein include determining the sequence of theentire region of the PI4K2B locus described herein as being of interest,e.g., between and including SNPs rs313548 and rs313567. In someembodiments, the methods described herein include determining thesequence of the entire region of the KCNIP4 locus described herein asbeing of interest, e.g., between and including SNPs rs6447982 andrs1364836. In some embodiments, the methods described herein includedetermining the sequence of the entire region of the CERK locusdescribed herein as being of interest, e.g., between and including SNPsrs801720 and rs710123. In some embodiments, the methods described hereininclude determining the sequence of the entire region of the SHANK3locus described herein as being of interest, e.g., between and includingSNPs rs713692 and rs756638. In some embodiments, the sequence isdetermined on both strands of DNA.

In order to detect polymorphisms and/or polymorphic variants, it willfrequently be desirable to amplify a portion of genomic DNA (gDNA)encompassing the polymorphic site. Such regions can be amplified andisolated by PCR using oligonucleotide primers designed based on genomicand/or cDNA sequences that flank the site. See e.g., PCR Primer: ALaboratory Manual, Dieffenbach and Dveksler, (Eds.); McPherson et al.,PCR Basics: From Background to Bench (Springer Verlag, 2000); Mattila etal., Nucleic Acids Res., 19:4967 (1991); Eckert et al., PCR Methods andApplications, 1:17 (1991); PCR (eds. McPherson et al., IRL Press,Oxford); and U.S. Pat. No. 4,683,202. Other amplification methods thatmay be employed include the ligase chain reaction (LCR) (Wu and Wallace,Genomics, 4:560 (1989), Landegren et al., Science, 241:1077 (1988),transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA,86:1173 (1989)), self-sustained sequence replication (Guatelli et al.,Proc. Nat. Acad. Sci. USA, 87:1874 (1990)), and nucleic acid basedsequence amplification (NASBA). Guidelines for selecting primers for PCRamplification are well known in the art. See, e.g., McPherson et al.,PCR Basics: From Background to Bench, Springer-Verlag, 2000. A varietyof computer programs for designing primers are available, e.g., ‘Oligo’(National Biosciences, Inc, Plymouth Minn.), MacVector (Kodak/IBI), andthe GCG suite of sequence analysis programs (Genetics Computer Group,Madison, Wis. 53711).

In one example, a sample (e.g., a sample comprising genomic DNA), isobtained from a subject. The DNA in the sample is then examined todetermine a haplotype as described herein. The haplotype can bedetermined by any method described herein, e.g., by sequencing or byhybridization of the gene in the genomic DNA, RNA, or cDNA to a nucleicacid probe, e.g., a DNA probe (which includes cDNA and oligonucleotideprobes) or an RNA probe. The nucleic acid probe can be designed tospecifically or preferentially hybridize with a particular polymorphicvariant.

In some embodiments, a peptide nucleic acid (PNA) probe can be usedinstead of a nucleic acid probe in the hybridization methods describedabove. PNA is a DNA mimetic with a peptide-like, inorganic backbone,e.g., N-(2-aminoethyl)glycine units, with an organic base (A, G, C, T orU) attached to the glycine nitrogen via a methylene carbonyl linker(see, e.g., Nielsen et al., Bioconjugate Chemistry, The AmericanChemical Society, 5:1 (1994)). The PNA probe can be designed tospecifically hybridize to a nucleic acid comprising a polymorphicvariant conferring susceptibility to or indicative of the presence ofSZ.

In some embodiments, restriction digest analysis can be used to detectthe existence of a polymorphic variant of a polymorphism, if alternatepolymorphic variants of the polymorphism result in the creation orelimination of a restriction site. A sample containing genomic DNA isobtained from the individual. Polymerase chain reaction (PCR) can beused to amplify a region comprising the polymorphic site, andrestriction fragment length polymorphism analysis is conducted (seeAusubel et al., Current Protocols in Molecular Biology, supra). Thedigestion pattern of the relevant DNA fragment indicates the presence orabsence of a particular polymorphic variant of the polymorphism and istherefore indicative of the presence or absence of susceptibility to SZ.

Sequence analysis can also be used to detect specific polymorphicvariants. A sample comprising DNA or RNA is obtained from the subject.PCR or other appropriate methods can be used to amplify a portionencompassing the polymorphic site, if desired. The sequence is thenascertained, using any standard method, and the presence of apolymorphic variant is determined.

Allele-specific oligonucleotides can also be used to detect the presenceof a polymorphic variant, e.g., through the use of dot-blothybridization of amplified oligonucleotides with allele-specificoligonucleotide (ASO) probes (see, for example, Saiki et al., Nature(London) 324:163-166 (1986)). An “allele-specific oligonucleotide” (alsoreferred to herein as an “allele-specific oligonucleotide probe”) istypically an oligonucleotide of approximately 10-50 base pairs,preferably approximately 15-30 base pairs, that specifically hybridizesto a nucleic acid region that contains a polymorphism. Anallele-specific oligonucleotide probe that is specific for particular apolymorphism can be prepared using standard methods (see Ausubel et al.,Current Protocols in Molecular Biology, supra).

Generally, to determine which of multiple polymorphic variants ispresent in a subject, a sample comprising DNA is obtained from theindividual. PCR can be used to amplify a portion encompassing thepolymorphic site. DNA containing the amplified portion may bedot-blotted, using standard methods (see Ausubel et al., CurrentProtocols in Molecular Biology, supra), and the blot contacted with theoligonucleotide probe. The presence of specific hybridization of theprobe to the DNA is then detected. Specific hybridization of anallele-specific oligonucleotide probe (specific for a polymorphicvariant indicative of susceptibility to SZ) to DNA from the subject isindicative of susceptibility to SZ.

In some embodiments, fluorescence polarization template-directeddye-terminator incorporation (FP-TDI) is used to determine which ofmultiple polymorphic variants of a polymorphism is present in a subject(Chen et al., (1999) Genome Research, 9(5):492-498). Rather thaninvolving use of allele-specific probes or primers, this method employsprimers that terminate adjacent to a polymorphic site, so that extensionof the primer by a single nucleotide results in incorporation of anucleotide complementary to the polymorphic variant at the polymorphicsite.

Real-time pyrophosphate DNA sequencing is yet another approach todetection of polymorphisms and polymorphic variants (Alderborn et al.,(2000) Genome Research, 10(8):1249-1258). Additional methods include,for example, PCR amplification in combination with denaturing highperformance liquid chromatography (dHPLC) (Underhill, P. A., et al.,Genome Research, Vol. 7, No. 10, pp. 996-1005, 1997).

The methods can include determining the genotype of a subject withrespect to both copies of the polymorphic site present in the genome.For example, the complete genotype may be characterized as −/−, as −/+,or as +/+, where a minus sign indicates the presence of the reference orwild type sequence at the polymorphic site, and the plus sign indicatesthe presence of a polymorphic variant other than the reference sequence.If multiple polymorphic variants exist at a site, this can beappropriately indicated by specifying which ones are present in thesubject. Any of the detection means described herein can be used todetermine the genotype of a subject with respect to one or both copiesof the polymorphism present in the subject's genome.

In some embodiments, it is desirable to employ methods that can detectthe presence of multiple polymorphisms (e.g., polymorphic variants at aplurality of polymorphic sites) in parallel or substantiallysimultaneously. Oligonucleotide arrays represent one suitable means fordoing so. Other methods, including methods in which reactions (e.g.,amplification, hybridization) are performed in individual vessels, e.g.,within individual wells of a multi-well plate or other vessel may alsobe performed so as to detect the presence of multiple polymorphicvariants (e.g., polymorphic variants at a plurality of polymorphicsites) in parallel or substantially simultaneously according to certainembodiments of the invention.

Probes

Nucleic acid probes can be used to detect and/or quantify the presenceof a particular target nucleic acid sequence within a sample of nucleicacid sequences, e.g., as hybridization probes, or to amplify aparticular target sequence within a sample, e.g., as a primer. Probeshave a complimentary nucleic acid sequence that selectively hybridizesto the target nucleic acid sequence. In order for a probe to hybridizeto a target sequence, the hybridization probe must have sufficientidentity with the target sequence, i.e., at least 70%, e.g., 80%, 90%,95%, 98% or more identity to the target sequence. The probe sequencemust also be sufficiently long so that the probe exhibits selectivityfor the target sequence over non-target sequences. For example, theprobe will be at least 20, e.g., 25, 30, 35, 50, 100, 200, 300, 400,500, 600, 700, 800, 900 or more, nucleotides in length. In someembodiments, the probes are not more than 30, 50, 100, 200, 300, 500,750, or 1000 nucleotides in length. Probes are typically about 20 toabout 1×10⁶ nucleotides in length. Probes include primers, whichgenerally refers to a single-stranded oligonucleotide probe that can actas a point of initiation of template-directed DNA synthesis usingmethods such as PCR (polymerase chain reaction), LCR (ligase chainreaction), etc., for amplification of a target sequence.

In some embodiments, the probe is a test probe, e.g., a probe that canbe used to detect polymorphisms in a region described herein, e.g.,polymorphisms as described herein. In some embodiments, the probe canhybridize to a target sequence within a region delimited by SNP rs313548and SNP rs313567 (described on the internet atncbi.nlm.nih.gov/SNP/snp_ref.cgi?rs=313548 andncbi.nlm.nih.gov/SNP/snp_ref.cgi?rs=313567, respectively). In someembodiments, the probe can hybridize to a target sequence within aregion delimited by SNP rs6447982 and SNP rs1364836 (described on theinternet at ncbi.nlm.nih.gov/SNP/snp_ref.cgi?rs=6447982 andncbi.nlm.nih.gov/SNP/snp_ref.cgi?rs=1364836, respectively). In someembodiments, the probe can hybridize to a target sequence within aregion delimited by SNP rs801720 and SNP rs710123 (described on theinternet at ncbi.nlm.nih.gov/SNP/snp_ref.cgi?rs=rs801720 andncbi.nlm.nih.gov/SNP/snp_ref.cgi?rs=rs710123, respectively). In someembodiments, the probe can hybridize to a target sequence within aregion delimited by SNP rs713692 and SNP rs756638 (described on theinternet at ncbi.nlm.nih.gov/SNP/snp_ref.cgi?rs=rs713692 andncbi.nlm.nih.gov/SNP/snp_ref.cgi?rs=rs756638, respectively).

In some embodiments, the probe can bind to another marker sequenceassociated with SZ, SPD or SD, as described herein.

Control probes can also be used. For example, a probe that binds a lessvariable sequence, e.g., repetitive DNA associated with a centromere ofa chromosome, can be used as a control. Probes that hybridize withvarious centromeric DNA and locus-specific DNA are availablecommercially, for example, from Vysis, Inc. (Downers Grove, Ill.),Molecular Probes, Inc. (Eugene, Oreg.), or from Cytocell (Oxfordshire,UK). Probe sets are available commercially, e.g., from AppliedBiosystems, e.g., the Assays-on-Demand SNP kits Alternatively, probescan be synthesized, e.g., chemically or in vitro, or made fromchromosomal or genomic DNA through standard techniques. For example,sources of DNA that can be used include genomic DNA, cloned DNAsequences, somatic cell hybrids that contain one, or a part of one,human chromosome along with the normal chromosome complement of thehost, and chromosomes purified by flow cytometry or microdissection. Theregion of interest can be isolated through cloning, or by site-specificamplification via the polymerase chain reaction (PCR). See, for example,Nath and Johnson, Biotechnic. Histochem., 1998, 73(1):6-22, Wheeless etal., Cytometry 1994, 17:319-326, and U.S. Pat. No. 5,491,224.

In some embodiments, the probes are labeled, e.g., by direct labeling,with a fluorophore, an organic molecule that fluoresces after absorbinglight of lower wavelength/higher energy. A directly labeled fluorophoreallows the probe to be visualized without a secondary detectionmolecule. After covalently attaching a fluorophore to a nucleotide, thenucleotide can be directly incorporated into the probe with standardtechniques such as nick translation, random priming, and PCR labeling.Alternatively, deoxycytidine nucleotides within the probe can betransaminated with a linker. The fluorophore then is covalently attachedto the transaminated deoxycytidine nucleotides. See, e.g., U.S. Pat. No.5,491,224.

Fluorophores of different colors can be chosen such that each probe in aset can be distinctly visualized. For example, a combination of thefollowing fluorophores can be used: 7-amino-4-methylcoumarin-3-aceticacid (AMCA), Texas Red™ (Molecular Probes, Inc., Eugene, Oreg.),5-(and-6)-carboxy-X-rhodamine, lissamine rhodamine B,5-(and-6)-carboxyfluorescein, fluorescein-5-isothiocyanate (FITC),7-diethylaminocoumarin-3-carboxylic acid,tetramethylrhodamine-5-(and-6)-isothiocyanate,5-(and-6)-carboxytetramethylrhodamine, 7-hydroxycoumarin-3-carboxylicacid, 6-[fluorescein 5-(and-6)-carboxamido]hexanoic acid,N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a diaza-3-indacenepropionicacid, eosin-5-isothiocyanate, erythrosin-5-isothiocyanate, and Cascade™blue acetylazide (Molecular Probes, Inc., Eugene, Oreg.). Fluorescentlylabeled probes can be viewed with a fluorescence microscope and anappropriate filter for each fluorophore, or by using dual or tripleband-pass filter sets to observe multiple fluorophores. See, forexample, U.S. Pat. No. 5,776,688. Alternatively, techniques such as flowcytometry can be used to examine the hybridization pattern of theprobes. Fluorescence-based arrays are also known in the art.

In other embodiments, the probes can be indirectly labeled with, e.g.,biotin or digoxygenin, or labeled with radioactive isotopes such as ³²Pand ³H. For example, a probe indirectly labeled with biotin can bedetected by avidin conjugated to a detectable marker. For example,avidin can be conjugated to an enzymatic marker such as alkalinephosphatase or horseradish peroxidase. Enzymatic markers can be detectedin standard colorimetric reactions using a substrate and/or a catalystfor the enzyme. Catalysts for alkaline phosphatase include5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium.Diaminobenzoate can be used as a catalyst for horseradish peroxidase.

Oligonucleotide probes that exhibit differential or selective binding topolymorphic sites may readily be designed by one of ordinary skill inthe art. For example, an oligonucleotide that is perfectly complementaryto a sequence that encompasses a polymorphic site (i.e., a sequence thatincludes the polymorphic site, within it or at one end) will generallyhybridize preferentially to a nucleic acid comprising that sequence, asopposed to a nucleic acid comprising an alternate polymorphic variant.

Arrays and Uses Thereof

In another aspect, the invention features arrays that include asubstrate having a plurality of addressable areas, and methods of usingthem. At least one area of the plurality includes a nucleic acid probethat binds specifically to a sequence comprising a polymorphism listedin Table A, and can be used to detect the absence or presence of saidpolymorphism, e.g., one or more SNPs, microsatellites, minisatellites,or indels, as described herein, to determine a haplotype. For example,the array can include one or more nucleic acid probes that can be usedto detect a polymorphism listed in Table A. In some embodiments, thearray further includes at least one area that includes a nucleic acidprobe that can be used to specifically detect another marker associatedwith SZ, SPD or SD, as described herein. The substrate can be, e.g., atwo-dimensional substrate known in the art such as a glass slide, awafer (e.g., silica or plastic), a mass spectroscopy plate, or athree-dimensional substrate such as a gel pad. In some embodiments, theprobes are nucleic acid capture probes.

Methods for generating arrays are known in the art and include, e.g.,photolithographic methods (see, e.g., U.S. Pat. Nos. 5,143,854;5,510,270; and 5,527,681), mechanical methods (e.g., directed-flowmethods as described in U.S. Pat. No. 5,384,261), pin-based methods(e.g., as described in U.S. Pat. No. 5,288,514), and bead-basedtechniques (e.g., as described in PCT US/93/04145). The array typicallyincludes oligonucleotide probes capable of specifically hybridizing todifferent polymorphic variants. According to the method, a nucleic acidof interest, e.g., a nucleic acid encompassing a polymorphic site,(which is typically amplified) is hybridized with the array and scanned.Hybridization and scanning are generally carried out according tostandard methods. See, e.g., Published PCT Application Nos. WO 92/10092and WO 95/11995, and U.S. Pat. No. 5,424,186. After hybridization andwashing, the array is scanned to determine the position on the array towhich the nucleic acid hybridizes. The hybridization data obtained fromthe scan is typically in the form of fluorescence intensities as afunction of location on the array.

Arrays can include multiple detection blocks (i.e., multiple groups ofprobes designed for detection of particular polymorphisms). Such arrayscan be used to analyze multiple different polymorphisms. Detectionblocks may be grouped within a single array or in multiple, separatearrays so that varying conditions (e.g., conditions optimized forparticular polymorphisms) may be used during the hybridization. Forexample, it may be desirable to provide for the detection of thosepolymorphisms that fall within G-C rich stretches of a genomic sequence,separately from those falling in A-T rich segments. Additionaldescription of use of oligonucleotide arrays for detection ofpolymorphisms can be found, for example, in U.S. Pat. Nos. 5,858,659 and5,837,832. In addition to oligonucleotide arrays, cDNA arrays may beused similarly in certain embodiments of the invention.

The methods described herein can include providing an array as describedherein; contacting the array with a sample, e.g., a portion of genomicDNA that includes at least a portion of human chromosome 4p and/or 22q,e.g., a region between SNP rs801720 and SNP rs710123, e.g., a regionbetween SNP rs713692 and rs756638, optionally, a different portion ofgenomic DNA, e.g., a portion that includes a different portion of humanchromosomes 22 and/or 4, or another chromosome, e.g., including anotherregion associated with SZ, SPD or SD., and detecting binding of anucleic acid from the sample to the array. Optionally, the methodincludes amplifying nucleic acid from the sample, e.g., genomic DNA thatincludes a portion of a human chromosome described herein, and,optionally, a region that includes another region associated with SZ,SPD, or SD, prior to or during contact with the array.

In some aspects, the methods described herein can include using an arraythat can ascertain differential expression patterns or copy numbers ofone or more genes in samples from normal and affected individuals (see,e.g., Redon et al., Nature. 444(7118):444-54 (2006)). For example,arrays of probes to a marker described herein can be used to measurepolymorphisms between DNA from a subject having SZ, SPD, or SD, andcontrol DNA, e.g., DNA obtained from an individual that does not haveSZ, SPD, or SD, and has no risk factors for SZ, SPD, or SD. Since theclones on the array contain sequence tags, their positions on the arrayare accurately known relative to the genomic sequence. Differenthybridization patterns between DNA from an individual afflicted with SZ,SPD or SD and DNA from a normal individual at areas in the arraycorresponding to markers in human chromosome 4p and/or 22q as describedherein, and, optionally, one or more other regions associated with SZ,SPD, or SD, are indicative of a risk of SZ. Methods for arrayproduction, hybridization, and analysis are described, e.g., in Snijderset al., (2001) Nat. Genetics 29:263-264; Klein et al., (1999) Proc.Natl. Acad. Sci. U.S.A. 96:4494-4499; Albertson et al., (2003) BreastCancer Research and Treatment 78:289-298; and Snijders et al. “BACmicroarray based comparative genomic hybridization.” In: Zhao et al.(eds), Bacterial Artificial Chromosomes: Methods and Protocols, Methodsin Molecular Biology, Humana Press, 2002. Real time quantitative PCR canalso be used to determine copy number.

In another aspect, the invention features methods of determining theabsence or presence of a haplotype associated with SZ as describedherein, using an array described above. The methods include providing atwo dimensional array having a plurality of addresses, each address ofthe plurality being positionally distinguishable from each other addressof the plurality having a unique nucleic acid capture probe, contactingthe array with a first sample from a test subject who is suspected ofhaving or being at risk for SZ, and comparing the binding of the firstsample with one or more references, e.g., binding of a sample from asubject who is known to have SZ, SPD, or SD, and/or binding of a samplefrom a subject who is unaffected, e.g., a control sample from a subjectwho neither has, nor has any risk factors for SZ, SPD, or SD. In someembodiments, the methods include contacting the array with a secondsample from a subject who has SZ, SPD or SD; and comparing the bindingof the first sample with the binding of the second sample. In someembodiments, the methods include contacting the array with a thirdsample from a cell or subject that does not have SZ and is not at riskfor SZ; and comparing the binding of the first sample with the bindingof the third sample. In some embodiments, the second and third samplesare from first or second-degree relatives of the test subject. Binding,e.g., in the case of a nucleic acid hybridization, with a capture probeat an address of the plurality, can be detected by any method known inthe art, e.g., by detection of a signal generated from a label attachedto the nucleic acid.

Schizophrenia, Schizotypal Personality Disorder, and SchizoaffectiveDisorder

The methods described herein can be used to determine an individual'srisk of developing schizophrenia (SZ), schizotypal personality disorder(SPD), and/or a schizoaffective disorder (SD).

Schizophrenia (SZ)

SZ is considered a clinical syndrome, and is probably a constellation ofseveral pathologies. Substantial heterogeneity is seen between cases,which is thought to reflect multiple overlapping etiologic factors,including both genetic and environmental contributions. A diagnosis ofSZ is typically indicated by chronic psychotic symptoms, e.g.,hallucinations and delusions. Disorganization of thought and behaviorare common and are considered distinguishing factors in the diagnosis ofSZ. Patients typically have some subtle impairments in cognition.Reduced emotional experience and expression, low drive, and impairedspeech are observed in a subgroup of patients. Cognitive, emotional andsocial impairments often appear early in life, while the psychoticsymptoms typically manifest in late adolescence or early adulthood inmen, a little later in women.

A diagnosis of SZ can be made according to the criteria reported in theDiagnostic and Statistical Manual of Mental Disorders, Fourth Edition,Text Revision, American Psychiatric Association, 2000, (referred toherein as DSM-IV) as follows:

Diagnostic Criteria for SZ

All six criteria must be met for a diagnosis of SZ.

A. Characteristic symptoms: Two (or more) of the following, each presentfor a significant portion of time during a one month period (or less ifsuccessfully treated):

(1) delusions

(2) hallucinations

(3) disorganized speech (e.g., frequent derailment or incoherence)

(4) grossly disorganized or catatonic behavior

(5) negative symptoms, e.g., affective flattening, alogia, or avolition

Only one criterion A symptom is required if delusions are bizarre orhallucinations consist of a voice keeping up a running commentary on theperson's behavior or thoughts, or two or more voices conversing witheach other.

B. Social/occupational dysfunction: For a significant portion of thetime since the onset of the disturbance, one or more major areas offunctioning such as work, interpersonal relations, or self-care aremarkedly below the level achieved prior to the onset (or when the onsetis in childhood or adolescence, failure to achieve expected level ofinterpersonal, academic, or occupational achievement).

C. Duration: Continuous signs of the disturbance persist for at least 6months. This 6-month period must include at least 1 month of symptoms(or less if successfully treated) that meet Criterion A (i.e.,active-phase symptoms) and may include periods of prodromal or residualsymptoms. During these prodromal or residual periods, the signs of thedisturbance may be manifested by only negative symptoms or two or moresymptoms listed in Criterion A present in an attenuated form (e.g., oddbeliefs, unusual perceptual experiences).

D. Schizoaffective and Mood Disorder Exclusion: Schizoaffective Disorderand Mood Disorder With Psychotic Features have been ruled out becauseeither (1) no major depressive, manic, or mixed episodes have occurredconcurrently with the active-phase symptoms; or (2) if mood episodeshave occurred during active-phase symptoms, their total duration hasbeen brief relative to the duration of the active and residual periods.

E. Substance/General Medical Condition Exclusion: The disturbance is notdue to the direct physiological effects of a substance (e.g., a drug ofabuse, a medication) or a general medical condition.

F. Relationship to a Pervasive Developmental Disorder: If the patienthas a history of Autistic Disorder or another Pervasive DevelopmentalDisorder, the additional diagnosis of SZ is made only if prominentdelusions or hallucinations are also present for at least a month (orless if successfully treated).

Schizoaffective Disorder (SD)

SD is characterized by the presence of affective (depressive or manic)symptoms and schizophrenic symptoms within the same, uninterruptedepisode of illness.

Diagnostic Criteria for Schizoaffective Disorder

The DSM-IV Criteria for a diagnosis of schizoaffective disorder is asfollows:

An uninterrupted period of illness during which, at some time, there iseither (1) a Major Depressive Episode (which must include depressedmood), (2) a Manic Episode, or (3) a Mixed Episode, concurrent withsymptoms that meet (4) Criterion A for SZ, above.

A. Criteria for Major Depressive Episode

At least five of the following symptoms must be present during the same2-week period and represent a change from previous functioning; at leastone of the symptoms is either (1) depressed mood or (2) loss of interestor pleasure.

(1) depressed mood most of the day, nearly every day, as indicated byeither subjective report (e.g., feels sad or empty) or observation madeby others (e.g., appears tearful). In children and adolescents, this canbe an irritable mood.

(2) markedly diminished interest or pleasure in all, or almost all,activities most of the day, nearly every day (as indicated by eithersubjective account or observation made by others)

(3) significant weight loss when not dieting or weight gain (e.g., achange of more than 5% of body weight in a month), or decrease orincrease in appetite nearly every day. (In children, failure to makeexpected weight gains is considered).

(4) insomnia or hypersomnia nearly every day

(5) psychomotor agitation or retardation nearly every day (observable byothers, not merely subjective feelings of restlessness or being sloweddown)

(6) fatigue or loss of energy nearly every day

(7) feelings of worthlessness or excessive or inappropriate guilt (whichmay be delusional) nearly every day (not merely self-reproach or guiltabout being sick)

(8) diminished ability to think or concentrate, or indecisiveness,nearly every day (either by subjective account or as observed by others)

(9) recurrent thoughts of death (not just fear of dying), recurrentsuicidal ideation without a specific plan, or a suicide attempt or aspecific plan for committing suicide

In addition, the symptoms do not meet criteria for a Mixed Episode. Thesymptoms cause clinically significant distress or impairment in social,occupational, or other important areas of functioning. The symptoms arenot due to the direct physiological effects of a substance (e.g., a drugof abuse, a medication) or a general medical condition (e.g.,hypothyroidism).

The symptoms are not better accounted for by Bereavement, i.e., afterthe loss of a loved one, the symptoms persist for longer than 2 months,or are characterized by marked functional impairment, morbidpreoccupation with worthlessness, suicidal ideation, psychotic symptoms,or psychomotor retardation.

B. Criteria for Manic Episode

A manic episode is a distinct period of abnormally and persistentlyelevated, expansive, or irritable mood, lasting at least one week (orany duration, if hospitalization is necessary).

During the period of mood disturbance, three (or more) of the followingsymptoms have persisted (four if the mood is only irritable) and havebeen present to a significant degree:

-   -   (1) inflated self-esteem or grandiosity    -   (2) decreased need for sleep (e.g., feels rested after only 3        hours of sleep)    -   (3) more talkative than usual or pressure to keep talking    -   (4) flight of ideas or subjective experience that thoughts are        racing    -   (5) distractibility (i.e., attention too easily drawn to        unimportant or irrelevant external stimuli)    -   (6) increase in goal-directed activity (either socially, at work        or school, or sexually) or psychomotor agitation    -   (7) excessive involvement in pleasurable activities that have a        high potential for painful consequences (e.g., engaging in        unrestrained buying sprees, sexual indiscretions, or foolish        business investments)

The symptoms do not meet criteria for a Mixed Episode. The mooddisturbance is sufficiently severe to cause marked impairment inoccupational functioning or in usual social activities or relationshipswith others, or to necessitate hospitalization to prevent harm to selfor others, or there are psychotic features. The symptoms are not due tothe direct physiological effects of a substance (e.g., a drug of abuse,a medication, or other treatment) or a general medical condition (e.g.,hyperthyroidism).

C. Criteria for Mixed Episode

A mixed episode occurs when the criteria are met both for a ManicEpisode and for a Major Depressive Episode (except for duration) nearlyevery day during at least a 1-week period. The mood disturbance issufficiently severe to cause marked impairment in occupationalfunctioning or in usual social activities or relationships with others,or to necessitate hospitalization to prevent harm to self or others, orthere are psychotic features.

The symptoms are not due to the direct physiological effects of asubstance (e.g., a drug of abuse, a medication, or other treatment) or ageneral medical condition (e.g., hyperthyroidism).

D. Criterion A of SZ

See above.

E. Types of SD

The type of SD may be may be specifiable, as either Bipolar Type, if thedisturbance includes a Manic or a Mixed Episode (or a Manic or a MixedEpisode and Major Depressive Episodes), or Depressive Type, if thedisturbance only includes Major

Depressive Episodes.

F. Associated Features

Features associated with SD include Learning Problems, Hypoactivity,Psychotic, Euphoric Mood, Depressed Mood, Somatic/Sexual Dysfunction,Hyperactivity, Guilt/Obsession, Odd/Eccentric/Suspicious Personality,Anxious/Fearful/Dependent Personality, and Dramatic/Erratic/AntisocialPersonality.

Schizotvpal Personality Disorder (SPD)

Diagnostic Criteria for SPD

A diagnosis of SPD under the criteria of the DSM-IV is generally basedon a pervasive pattern of social and interpersonal deficits marked byacute discomfort with, and reduced capacity for, close relationships aswell as by cognitive or perceptual distortions and eccentricities ofbehavior, beginning by early adulthood and present in a variety ofcontexts, as indicated by five (or more) of the following:

(1) ideas of reference (excluding delusions of reference)

(2) odd beliefs or magical thinking that influences behavior and is

(3) inconsistent with subcultural norms (e.g., superstitiousness, beliefin clairvoyance, telepathy, or “sixth sense;” in children andadolescents, bizarre fantasies or preoccupations)

(4) unusual perceptual experiences, including bodily illusions

(5) odd thinking and speech (e.g., vague, circumstantial, metaphorical,overelaborate, or stereotyped)

(6) suspiciousness or paranoid ideation

(7) inappropriate or constricted affect

(8) behavior or appearance that is odd, eccentric, or peculiar

(9) lack of close friends or confidants other than first-degreerelatives

(10) excessive social anxiety that does not diminish with familiarityand tends to be associated with paranoid fears rather than negativejudgments about self

SPD is diagnosed if the symptoms do not occur exclusively during thecourse of SZ, a Mood Disorder With Psychotic Features, another PsychoticDisorder, or a Pervasive Developmental Disorder, and the disturbance isnot due to the direct physiological effects of a substance (e.g., a drugof abuse, a medication) or a general medical condition.

Associated features of SPD include Depressed Mood andOdd/Eccentric/Suspicious Personality.

Endophenotypes in SZ

A number of endophenotypes, i.e., intermediate phenotypes, that may moreclosely reflect biological mechanisms behind SZ, have been suggested,such as prepulse inhibition, structural abnormalities evident in MRIscans, specific domains of cognition (e.g., executive function), finemotor performance, working memory, etc.

Endophenotypes can also include clinical manifestations such ashallucinations, paranoia, mania, depression, obsessive-compulsivesymptoms, etc., as well as response or lack of response to drugs andcomorbidity for substance and alcohol abuse.

See, e.g., Kendler et al., Am J Psychiatry 152(5):749-54 (1995);Gottesman and Gould, Am J Psychiatry 160(4):636-45 (2003); Cadenhead,Psychiatric Clinics of North America. 25(4):837-53 (2002); Gottesman andGould, American Journal of Psychiatry. 160(4):636-45 (2003); Heinrichs,Neuroscience & Biobehavioral Reviews. 28(4):379-94 (2004); and Zobel andMaier, Nervenarzt. 75(3):205-14 (2004).

There is now evidence that some candidate genes that were identifiedusing DSM-IV type categorical definitions for “affected” individuals mayinfluence specific endophenotypes, see, e.g., Baker et al., BiolPsychiatry 58(1):23-31 (2005); Cannon et al., Arch Gen Psychiatry62(11):1205-13 (2005); Gothelf et al., Nat Neurosci 8(11):1500-2 (2005);Hallmayer et al., Am J Hum Genet 77(3):468-76 (2005); Callicott et al.,Proc Natl Acad Sci USA 102(24):8627-32 (2005); Gornick et al., J AutismDev Disord 1-8 (2005). Thus, the methods described herein can be used toassociate haplotypes of 22q13 with specific endophenotypes.

Current Treatment of SZ, SD, or SPD

Subjects with SZ typically require acute treatment for psychoticexacerbations, and long-term treatment including maintenance andprophylactic strategies to sustain symptom improvement and preventrecurrence of psychosis. Subjects with schizoaffective disorderexperience the symptoms of both SZ and affective disorder (manic and/ordepressive), thus require the specific treatments for each disorder.Subjects with SPD sometimes require medication for acute psychoticepisodes but are often treated using psychosocial methods. The methodsdescribed herein can include the administration of one or more acceptedor experimental treatment modalities to a person identified as at riskof developing SZ, SPD, or a SD, based on the presence of a haplotypeassociated with SZ, SPD, or SD. Currently accepted treatments presentlyinclude both pharmacologic and psychosocial management, and occasionallyelectroconvulsive therapy (ECT).

Standard pharmacologic therapies for SZ and SD include theadministration of one or more antipsychotic medications, which aretypically antagonists acting at postsynaptic D₂ dopamine receptors inthe brain. Antipsychotic medications include conventional, or firstgeneration, antipsychotic agents, which are sometimes referred to asneuroleptics because of their neurologic side effects, and secondgeneration antipsychotic agents, which are less likely to exhibitneuroleptic effects and have been termed atypical antipsychotics.

In some embodiments, the methods described herein include theadministration of one or more antipsychotic medications to a personidentified by a method described herein as being at risk of developingSZ, SPD, or SD. Antipsychotic medications substantially reduce the riskof relapse in the stable phase of illness. In some embodiments, themethods include the administration of a first generation antipsychoticmedication at a dose that is around the “extrapyramidal symptom (EPS)threshold” (i.e., the dose that will induce extrapyramidal side effects,e.g., bradykinesia, rigidity, or dyskinesia, with minimal rigiditydetectable on physical examination, and/or a second-generationantipsychotics at a dose that is therapeutic, yet below the EPSthreshold.

Standard pharmacologic therapies for SD also include the administrationof a combination of antidepressant, and anti-anxiety medication.Suitable antidepressants include serotonergic antidepressants, e.g.,fluoxetine or trazodone. Suitable anxiolytics include benzodiazepines,e.g., lorazepam, clonazepam. Lithium can also be administered. Thus, insome embodiments, the methods can include the administration of one ormore antidepressant and/or anti-anxiety medications to a personidentified as at risk of developing SZ, SPD, or SD.

The methods can also include psychosocial and rehabilitationinterventions, e.g., interventions that are generally accepted astherapeutically beneficial, e.g., cognitive-behavioral therapy fortreatment-resistant positive psychotic symptoms; supportive,problem-solving, educationally oriented psychotherapy; family therapyand education programs aimed at helping patients and their familiesunderstand the patient's illness, reduce stress, and enhance copingcapabilities; social and living skills training; supported employmentprograms; and/or the provision of supervised residential livingarrangements.

Currently accepted treatments for SZ are described in greater detail inthe Practice Guideline for the Treatment of Patients With SchizophreniaAmerican Psychiatric Association, Second Edition, American PsychiatricAssociation, 2004, which is incorporated herein by reference in itsentirety.

Methods of Determining Treatment Regimens and Methods of Treating SZ,SPD or SD

As described herein, the presence of haplotypes described herein hasbeen correlated with an increased risk of developing or having SZ, SPD,or SD. Thus, the new methods can also include selecting a treatmentregimen for a subject determined to be at risk for developing SZ, SPD orSD, based upon the absence or presence of a haplotype associated with SZas described herein. The determination of a treatment regimen can alsobe based upon the absence or presence of other risk factors associatedwith SZ, e.g., as described herein. Therefore, the methods of theinvention can include selecting a treatment regimen for a subject havingone or more risk factors for SZ, and having a haplotype describedherein. The methods can also include administering a treatment regimento a subject having, or at risk for developing, SZ to thereby treat,prevent or delay further progression of the disease. A treatment regimencan include the administration of antipsychotic medications to a subjectidentified as at risk of developing SZ before the onset of any psychoticepisodes.

As used herein, the term “treat” or “treatment” is defined as theapplication or administration of a treatment regimen, e.g., atherapeutic agent or modality, to a subject, e.g., a patient. Thesubject can be a patient having SZ, a symptom of SZ or at risk ofdeveloping (i.e., a predisposition toward) SZ. The treatment can be tocure, heal, alleviate, relieve, alter, remedy, ameliorate, palliate,improve or affect SZ, the symptoms of SZ or the predisposition towardSZ.

The methods of the invention, e.g., methods of determining a treatmentregimen and methods of treatment or prevention of SZ, can furtherinclude the step of monitoring the subject, e.g., for a change (e.g., anincrease or decrease) in one or more of the diagnostic criteria for SZlisted herein, or any other parameter related to clinical outcome. Thesubject can be monitored in one or more of the following periods: priorto beginning of treatment; during the treatment; or after one or moreelements of the treatment have been administered. Monitoring can be usedto evaluate the need for further treatment with the same or a differenttherapeutic agent or modality. Generally, a decrease in one or more ofthe parameters described above is indicative of the improved conditionof the subject, although with red blood cell and platelet levels, anincrease can be associated with the improved condition of the subject.

The methods can be used, e.g., to evaluate the suitability of, or tochoose between alternative treatments, e.g., a particular dosage, modeof delivery, time of delivery, inclusion of adjunctive therapy, e.g.,administration in combination with a second agent, or generally todetermine the subject's probable drug response genotype. In a preferredembodiment, a treatment for SZ can be evaluated by administering thesame treatment or combinations or treatments to a subject having SZ, SPDor SD and a haplotype as described herein and to a subject that has SZbut does not have a haplotype as described herein. The effects of thetreatment or combination of treatments on each of these subjects can beused to determine if a treatment or combination of treatments isparticularly effective on a sub-group of subjects having SZ, SPD or SD.In other embodiments, various treatments or combinations of treatmentscan be evaluated by administering two different treatments orcombinations of treatments to at least two different subjects having SZ,SPD or SD and a haplotype as described herein. Such methods can be usedto determine if a particular treatment or combination of treatments ismore effective than others in treating this subset of SZ, SPD and/or SDpatients.

Various treatment regimens are known for treating SZ, e.g., as describedherein.

Pharmacogenomics

With regards to both prophylactic and therapeutic methods of treatmentof SZ, such treatments may be specifically tailored or modified, basedon knowledge obtained from the field of pharmacogenomics.“Pharmacogenomics,” as used herein, refers to the application ofgenomics technologies such as structural chromosomal analysis, to drugsin clinical development and on the market. See, for example, Eichelbaumet al., Clin. Exp. Pharmacol. Physiol. 23:983-985 (1996) and Linder etal., Clin. Chem. 43:254-266 (1997). Specifically, as used herein, theterm refers the study of how a patient's genes determine his or herresponse to a drug (e.g., a patient's “drug response phenotype,” or“drug response genotype”). Thus, another aspect of the inventionprovides methods for tailoring an individual's prophylactic ortherapeutic treatment according to that individual's drug responsegenotype.

Information generated from pharmacogenomic research using a methoddescribed herein can be used to determine appropriate dosage andtreatment regimens for prophylactic or therapeutic treatment of anindividual. This knowledge, when applied to dosing or drug selection,can avoid adverse reactions or therapeutic failure and thus enhancetherapeutic or prophylactic efficiency when administering a therapeuticcomposition, e.g., a cytotoxic agent or combination of cytotoxic agents,to a patient, as a means of treating or preventing SZ.

In one embodiment, a physician or clinician may consider applyingknowledge obtained in relevant pharmacogenomics studies, e.g., using amethod described herein, when determining whether to administer apharmaceutical composition, e.g., an antipsychotic agent or acombination of antipsychotic agents, to a subject. In anotherembodiment, a physician or clinician may consider applying suchknowledge when determining the dosage, e.g., amount per treatment orfrequency of treatments, of a treatment, e.g., a antipsychotic agent orcombination of antipsychotic agents, administered to a patient.

As one example, a physician or clinician may determine (or havedetermined, e.g., by a laboratory) the haplotype of a subject asdescribed herein, and optionally one or more other markers associatedwith SZ, SPD, or SD, of one or a group of subjects who may beparticipating in a clinical trial, wherein the subjects have SZ, SPD, orSD, and the clinical trial is designed to test the efficacy of apharmaceutical composition, e.g., an antipsychotic or combination ofantipsychotic agents, and wherein the physician or clinician attempts tocorrelate the genotypes of the subjects with their response to thepharmaceutical composition.

As another example, information regarding a haplotype associated with anincreased risk of SZ, SPD or SD, as described herein, can be used tostratify or select a subject population for a clinical trial. Theinformation can, in some embodiments, be used to stratify individualsthat may exhibit a toxic response to a treatment from those that willnot. In other cases, the information can be used to separate those thatwill be non-responders from those who will be responders. The haplotypesdescribed herein can be used in pharmacogenomics-based design and managethe conduct of a clinical trial, e.g., as described in U.S. Pat. Pub.No. 2003/0108938.

As another example, information regarding a haplotype associated with anincreased risk of SZ, SPD or SD, as described herein, can be used tostratify or select human cells or cell lines for drug testing purposes.Human cells are useful for studying the effect of a polymorphism onphysiological function, and for identifying and/or evaluating potentialtherapeutic agents for the treatment of SZ, SPD, or SD, e.g.,antipsychotics. Thus the methods can include performing the presentmethods on genetic material from a cell line. The information can, insome embodiments, be used to separate cells that respond particulardrugs from those that do not respond, e.g. which cells show alteredsecond messenger signaling.

Theranostics

Also included herein are compositions and methods for the identificationand treatment of subjects who have an increased risk of SZ, SPD or SD,such that a theranostic approach can be taken to test such individualsto determine the effectiveness of a particular therapeutic intervention(e.g., a pharmaceutical or non-pharmaceutical intervention as describedherein) and to alter the intervention to 1) reduce the risk ofdeveloping adverse outcomes and 2) enhance the effectiveness of theintervention. Thus, in addition to diagnosing or confirming thepredisposition to SZ, SPD or SD, the methods and compositions describedherein also provide a means of optimizing the treatment of a subjecthaving such a disorder. Provided herein is a theranostic approach totreating and preventing SZ, SPD or SD, by integrating diagnostics andtherapeutics to improve the real-time treatment of a subject.Practically, this means creating tests that can identify which patientsare most suited to a particular therapy, and providing feedback on howwell a drug is working to optimize treatment regimens.

Within the clinical trial setting, a theranostic method or compositionof the invention can provide key information to optimize trial design,monitor efficacy, and enhance drug safety. For instance, “trial design”theranostics can be used for patient stratification, determination ofpatient eligibility (inclusion/exclusion), creation of homogeneoustreatment groups, and selection of patient samples that arerepresentative of the general population. Such theranostic tests cantherefore provide the means for patient efficacy enrichment, therebyminimizing the number of individuals needed for trial recruitment.“Efficacy” theranostics are useful for monitoring therapy and assessingefficacy criteria. Finally, “safety” theranostics can be used to preventadverse drug reactions or avoid medication error.

The methods described herein can include retrospective analysis ofclinical trial data as well, both at the subject level and for theentire trial, to detect correlations between a haplotype as describedherein and any measurable or quantifiable parameter relating to theoutcome of the treatment, e.g., efficacy (the results of which may bebinary (i.e., yes and no) as well as along a continuum), side-effectprofile (e.g., weight gain, metabolic dysfunction, lipid dysfunction,movement disorders, or extrapyramidal symptoms), treatment maintenanceand discontinuation rates, return to work status, hospitalizations,suicidality, total healthcare cost, social functioning scales, responseto non-pharmacological treatments, and/or dose response curves. Theresults of these correlations can then be used to influencedecision-making, e.g., regarding treatment or therapeutic strategies,provision of services, and/or payment. For example, a correlationbetween a positive outcome parameter (e.g., high efficacy, low sideeffect profile, high treatment maintenance/low discontinuation rates,good return to work status, low hospitalizations, low suicidality, lowtotal healthcare cost, high social function scale, favorable response tonon-pharmacological treatments, and/or acceptable dose response curves)and a selected haplotype can influence treatment such that the treatmentis recommended or selected for a subject having the selected haplotype.

Kits

Also within the scope of the invention are kits comprising a probe thathybridizes with a region of human chromosome as described herein and canbe used to detect a polymorphism described herein. The kit can includeone or more other elements including: instructions for use; and otherreagents, e.g., a label, or an agent useful for attaching a label to theprobe. Instructions for use can include instructions for diagnosticapplications of the probe for assessing risk of SZ in a method describedherein. Other instructions can include instructions for attaching alabel to the probe, instructions for performing in situ analysis withthe probe, and/or instructions for obtaining a sample to be analyzedfrom a subject. As discussed above, the kit can include a label, e.g.,any of the labels described herein. In some embodiments, the kitincludes a labeled probe that hybridizes to a region of human chromosomeas described herein, e.g., a labeled probe as described herein.

The kit can also include one or more additional probes that hybridize tothe same chromosome, e.g., chromosome 4 or 22, or another chromosome orportion thereof that can have an abnormality associated with risk forSZ. For example, the additional probe or probes can be: a probe thathybridizes to human chromosome 22q11-12 or a portion thereof, (e.g., aprobe that detects a sequence associated with SZ in this region ofchromosome 22), or probes that hybridize to all or a portion of 22q12.3(e.g., near D22S283), 22q11.2, 22q11.2, 22q11-q13, 1q42.1, 1q42.1, 18p,15q15, 14q32.3, 13q34, 13q32, 12q24, 11q14-q21, 1q21-q22, 10p15-p13(e.g., near D10S189), 10q22.3, 8p21, 6q13-q26, 6p22.3, 6p23,5q11.2-q13.3, and/or 3p25. A kit that includes additional probes canfurther include labels, e.g., one or more of the same or differentlabels for the probes. In other embodiments, the additional probe orprobes provided with the kit can be a labeled probe or probes. When thekit further includes one or more additional probe or probes, the kit canfurther provide instructions for the use of the additional probe orprobes.

Kits for use in self-testing can also be provided. For example, suchtest kits can include devices and instructions that a subject can use toobtain a sample, e.g., of buccal cells or blood, without the aid of ahealth care provider. For example, buccal cells can be obtained using abuccal swab or brush, or using mouthwash.

Kits as provided herein can also include a mailer, e.g., a postage paidenvelope or mailing pack, that can be used to return the sample foranalysis, e.g., to a laboratory. The kit can include one or morecontainers for the sample, or the sample can be in a standard bloodcollection vial. The kit can also include one or more of an informedconsent form, a test requisition form, and instructions on how to usethe kit in a method described herein. Methods for using such kits arealso included herein. One or more of the forms, e.g., the testrequisition form, and the container holding the sample, can be coded,e.g., with a bar code, for identifying the subject who provided thesample.

Databases

Also provided herein are databases that include a list of polymorphismsas described herein, and wherein the list is largely or entirely limitedto polymorphisms identified as useful in performing genetic diagnosis ofor determination of susceptibility to SZ, SPD or SD as described herein.The list is stored, e.g., on a flat file or computer-readable medium.The databases can further include information regarding one or moresubjects, e.g., whether a subject is affected or unaffected, clinicalinformation such as endophenotype, age of onset of symptoms, anytreatments administered and outcomes (e.g., data relevant topharmacogenomics, diagnostics or theranostics), and other details, e.g.,about the disorder in the subject, or environmental or other geneticfactors. The databases can be used to detect correlations between aparticular haplotype and the information regarding the subject, e.g., todetect correlations between a haplotype and a particular endophenotype,or treatment response.

Engineered Cells

Also provided herein are engineered cells that harbor one or morepolymorphism described herein, e.g., one or more polymorphisms thatconstitute a haplotype associated with SZ, SPD, or SD. Such cells areuseful for studying the effect of a polymorphism on physiologicalfunction, and for identifying and/or evaluating potential therapeuticagents for the treatment of SZ, SPD, or SD, e.g., antipsychotics.

As one example, included herein are cells in which one of the variousalleles of the genes described herein has be re-created that isassociated with an increased risk of SZ, SD, or SPD. Methods are knownin the art for generating cells, e.g., by homologous recombinationbetween the endogenous gene and an exogenous DNA molecule introducedinto a cell, e.g., a cell of an animal. In some embodiments, the cellscan be used to generate transgenic animals using methods known in theart. The cells are preferably mammalian cells, e.g., neuronal typecells, in which an endogenous gene has been altered to include apolymorphism as described herein. Techniques such as targeted homologousrecombinations, can be used to insert the heterologous DNA as describedin, e.g., Chappel, U.S. Pat. No. 5,272,071; WO 91/06667, published inMay 16, 1991.

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

EXAMPLES Example 1 Whole Autosomal Screen for Quantitative Trait Loci(QTLs) Influencing Adult Schizotypy

640 adult offspring and their parents in 165 families who haveparticipated in the longitudinal Louisville Twin Study of behavioraldevelopment were recruited and their informed consent to participate inthis project was obtained. The offspring form 1,150 twin and siblingpairs. The sample includes 21 dizygotic (DZ, i.e., fraternal) male, and21 opposite-sexed twin pairs; 58 monozygotic (MZ, i.e., identical)female and 21 MZ male pairs; and 316 female, 215 male and 467 oppositesibling pairs. MZ twin pairs do not aid in linkage detection but permitmonitoring of estimates of shared environmental and residual additivegenetic variance.

The MMPI-2 is the slightly revised form of a well-verified personalityquestionnaire that has been widely used for decades in research andclinical settings. The basic clinical scales were created todifferentiate empirically between diagnosed persons and controls(Hathaway and McKinley (1989), Manual for the MINNESOTA MULTIPHASICPERSONALITY INVENTORY-2™ (MMPI-2™) Minneapolis, University of MinnesotaPress). In the present study, MMPI-2 questionnaires were scored andscaled by computer, using procedures and tables provided by Hathaway andMcKinley (Hathaway and McKinley (1989), supra), including theK-correction for defensive responding. The whole autosomal screen wasperformed on standardized, untransformed MMPI-2 scaled scores.Subsequently, the scores were transformed using the natural log toreduce the influence of outlying scores on the results. Regressionprocedures are generally quite robust with respect to non-normality, butthe influence of outliers must be evaluated. For all scales thelog-transform was sufficient to meet the criteria of no probableoutliers in the standard box plot procedure as implemented in Minitabstatistical software (Minitab, Inc.).

Genotypes were determined at 227 polymorphic markers (meanheterozygosity 0.81) with an average spacing of 16.2 cM. We usedstandardized procedures for fluorescently-labeled primers (AppliedBiosystems, approximately 85% of genotypes) or ³²P-labeled primers(Scored on a Molecular Dynamics phosphoimager, 15% of genotypes) asdescribed previously (Brennan et al., (2000) Genomics 63: 430-432).Genetic maps were constructed using MultiMap (Matise et al., (1994) Nat.Genet. 6: 384-390). The genetic map derived for chromosome 4 markers isshown in Table 1 and the map for chromosome 22 markers, was aspreviously described (Brennan et al., (2000), supra).

TABLE 1 Microsatellite Markers for Chromosome 4 Kosambi cM Marker MB^(b)Female Male Sex Averaged D4s126 30.22495 0 0 0 D4s1599^(a) NA 27.4 14.120.1 D4s391 27.221546 57.2 33.5 43.6 D4s174 40.528682 76.3 43.8 57.9D4s1645 61.665571 103.2 47.6 71.1 D4s423 92.691891 158 67.2 102 D4s406111.93789 177 80.8 118.5 D4s402 120.36763 187.7 86.1 126.4 D4sIL-2123.53890 192.5 89.3 130.5 D4s175 139.58611 212.8 94.2 142.2 FGA(UniSTS: 156198) 155.72822 232.9 105.5 157.2 D4s1636 166.73249 251.7112.6 169.2 D4s1554 184.92558 287.6 131.4 194.7 D4s2930 190.33384 300.9152.4 211.3 Footnotes Table 1: ^(a)Not placed on physical map.^(b)Genome Build 36.2

Proportions identical by descent (IBD) were estimated for sibling and DZtwin pairs at 3,343 1-cM points across the 22 autosomes usingMAPMAKER/Sib (Kruglyak and Lander, (1995) Am. J. Hum. Genet. 57:439-454). Linkage analyses were performed using a multiple regressionprocedure (Fulker et al., (1995) Am. J. Hum. Genet. 56: 1224-1233:P1_(i) =b ₀ +b ₁ P2_(i) +b ₂π_(ij) +b ₃ p2_(i)π_(iR) +b ₄π_(iR) +b ₅P2_(i)π_(iR)

Here P1_(i) and P2_(i) are the personality scores of the ith sibling ortwin pair; π_(ij) is the estimated proportion of chromosomal materialIBD for the ith pair at the jth 1-cM point on a chromosome; the π_(iR)is the overall coefficient of relationship of the ith pair, 0.5 for DZtwins and sibling pairs and 1.0 for MZ pairs. For each chromosomalpoint, the t-value for b₃ was evaluated for evidence of linkage.One-tailed probabilities were calculated from the normal distribution,which applies in large samples.

PCR Amplification and Genotyping

Microsatellite markers were genotyped following standard procedures with10 ng genomic DNA in 10 μl reaction volumes, using PCR reagents obtainedfrom Applied Biosystems (ABI, Foster City, Calif.), with standardreaction conditions as described previously (Brennan et al., (2000)Genomics 63, 430-432; Brennan and Condra, (2005) Am. J. Med Genet. BNeuropsychiatr. Genet. 139, 69-72). Fragments were analyzed using an ABIPRISM 377 DNA sequencer, with GeneScan and Genotyper software packagesfollowed by manual confirmation.

Genetic Analysis

Mendelian inheritance for all markers was confirmed using the GENEHUNTERgenetic analysis software (Version 2.0; Kruglyak et al., (1996) Am. J.Hum. Genet. 58, 1347-1363). Following initial analysis, putativelyrecombinant chromosomes were identified to detect possible genotypingerrors, and genotyping was repeated to confirm recombination events. Asa reference genetic map, a map previously based upon approximately 1000informative meioses for chromosome 22q (Brennan et al., (2000), supra)was used. Input allele frequencies for microsatellite markers were theempirical frequencies determined for approximately 550 unrelatedindividuals from the Louisville metropolitan area (Brennan et al.,(2000), supra).

Analysis of the linkage t-value across the 22 autosomes for the MMPI-2schizophrenia scale revealed two major peaks. There was a significantlinkages on 4p15.1 spanning D4S391 (t=4.34, P=7×10⁻⁶), at 41-45 cM fromp-ter (FIG. 1). Chromosome 22q had two peaks, a major peak on 22q13.33at 63 cM, about midway between markers D22S526 and D22S1744 (t=3.83,P=6×10⁻⁵), located at 61.8 and 64.6 cM from the p-ter respectively, anda somewhat lower peak located at 52 cM (t=3.34, P=4.2×10⁻⁴) (FIG. 2).

The MMPI-2 validity K-scale was tested in both the 4p15 and 22q13regions. The K-scale is used to correct five of the basic clinicalscales for defensive responding, including the schizophrenia andpsychasthenia scales (Hathaway and McKinley (1989), supra). There was noindication of linkage for the K-scale in either region, indicating thatthe K-correction was not a source for the linkages.

Example 2 Chromosome 4p Detailed Studies by QTL Linkage Analysis forMMP-Scales

The findings on chromosome 4 were explored with eight other MMPI-2 basicclinical scales; the results are shown in Table 2.

TABLE 2 MMPI-2 QTL linkages on Chromosome 4p for Basic Clinical ScalesLinkage Peak cM MMPI-2 Scale t value P value 41-45 schizophrenia(schizotypy) 4.34 0.000007 40-41 psychasthenia (obsessive- 3.83 0.000063compulsive) 43 psychopathic deviance 3.17 0.00076 39 Hysteria 2.840.0022 47 hypochondriasis 2.59 0.0048 44 depression 2.26 0.012 40 mania2.22 0.013

The psychasthenia scale (Hathaway and McKinley (1989), Manual for theMINNESOTA MULTIPHASIC PERSONALITY INVENTORY-2™ (MMPI-2™) Minneapolis,University of Minnesota Press) a measure of obsessive-compulsiveness,showed linkage (t=3.83, P=6×10⁻⁵) in this region, and five other basicclinical scales—hysteria, psychopathic deviance, hypochondriasis,depression and mania (Hathaway and McKinley (1989), supra)—were elevatedin this region as well. A total of seven of the basic clinical scales ofthe MMPI-2, thus, formed a striking, nearly uniform pattern of elevationnear D4S391. Although the MMPI-2 scales are correlated, this does notaccount for the pattern found on 4p because the scales show scatteredpatterns seen for these linkages elsewhere in the QTL scan.

The 4p15 region was also probed with additional scales and subscales:the six Harris-Lingoes schizophrenia subscales (Hathaway and McKinley(1989), supra; Butcher et al., (1989) Development and use of the MMPI-2content scales. University of Minnesota Press, Minneapolis; the contentscales (Hathaway and McKinley (1989), supra) for obsessiveness, fearsand bizarre mentation; and the ten Weiner-Harmonl subtle vs. obvioussubscales. Six of the subscales showed linkage in this region, andinterestingly, their peaks only partially coincided with those forschizophrenia and psychasthenia (Table 3).

TABLE 3 MMPI-2 QTL linkages on Chromosome 4p for Subscales Linkage PeakcM MMPI-2 Scale t value P value 43-44 paranoia obvious 3.46 0.00027 39hysteria obvious 3.99 0.000033 39-41 anxiety 3.20 0.00068 34obsessiveness 4.72 0.0000012 31 Sc6 subscale (bizarre sensory) 4.300.0000085 29 Sc5 subscale (lack of ego 3.39 0.00034 mastery, defectiveinhibition)

The paranoia obvious subscale showed linkage (t=3.46, P=2.7×10⁻⁴) at43-44 cM, the peak area for the schizophrenia scale. The hysteriaobvious subscale showed linkage (t=3.99, P=3.3×10⁻⁵) at 39 cM, near thepeak for the psychasthenia scale, as did the anxiety scale (t=3.2,P=7×10⁻⁴) at 39-41 cM. The obsessiveness content scale revealed a highlysignificant linkage (t=4.72, P=1.2×10⁻⁶) at 34 cM, which is five cMdistal to the peak for psychasthenia. The schizophrenia Sc6 subscale, ameasure of bizarre sensory experiences, revealed a significant linkage(t=4.30, P=8.6×10⁻⁶) at 31 cM, and the schizophrenia Sc5 subscale, ameasure of lack of ego mastery and defective inhibition, gave a peak at29 cM (t=3.39, P=3.4×10⁻⁴).

These results suggest that there are at least two separable QTLsinfluencing aspects of schizotypy (e.g., schizophrenia susceptibility)and related personality and psychopathology in the 4p15 region. Logtransformation had little effect, slightly reducing the peak linkaget=values from 4.34 to 4.10 for schizophrenia and from 3.83 to 3.44 forpsychasthenia and without changing the locations of the maximums (notshown). This indicates that the influence of extreme scores on theseresults is small.

Example 3 Chromosome 4p Gene Confirmation by TDT in Clinical Samples

Samples from thirty-nine families, comprising 212 individuals, eachhaving multiple affected siblings were obtained from NIMH.Self-description of heritage was as follows: African-American, 14families; European/Mediterranean, 25 families. DSM-IIIR or DSM-IVcriteria were compiled for all subjects by researchers at ColumbiaUniversity, Harvard University and Washington University. Detailedinformation on ascertainment, diagnosis and informed consent has beenpreviously provided by these groups (Cloninger et al., (1998) Am. J.Med. Genet. 81, 275-281; Faraone et al., (1998) Am. J. Med. Genet. 81,290-295; Kaufmann et al., (1998) Am. J. Med. Genet. 81, 282-289).

Using the DSM-IIIR/IV criteria for SZ, the sample contained 51 affectedsibling pairs, and using a broadest disease definition that includedschizotypal personality disorder and schizoaffective disorder, thesample contained 91 affected sibling pairs.

SNPs were genotyped by ABI ASSAYS-ON-DEMAND™ genotyping kits using theconditions suggested by the supplier (5 μA reactions in 384-well plates,containing 4.5 ng genomic DNA). PCR products were analyzed using the ABIPrism 7900HT Sequence Detection System. In cases where a reaction failed(<3% of total), or the results were not consistent with Mendelianinheritance (<0.5% of total), a second reaction was carried out toresolve discrepancies.

Transmission disequilibrium (TDT) analysis was performed to test for thepossibility of allelic association in the presence linkage (Laird andLange, (2006) Nat. Rev. Genet. 7, 385-394). TDT analysis was performedusing TRANSMIT (Version 2.5.2), which uses a robust variance estimatethat allows for multiple affected members in each family, in effect,treating families, rather than siblings, as independent entities(Clayton, (1999) Am. J. Hum. Genet. 65, 1170-1177; Martin et al., (2003)Am. J. Hum. Genet. 73, 1016-1026). Alleles were aggregated so as toprevent elevation of X² values that can arise due to expectations forrare haplotypes. The resulting global X² analyses estimate thesignificance of the transmission distribution for all alleles combined,with rare haplotypes being treated as a single group. Similarly, X²values for transmission of individual alleles, with one degree offreedom, were determined by TRANSMIT. We used two approaches to arriveat conservative estimates of Type I error probabilities for TDTanalysis. First, Bonferroni corrections for multiple comparisons wereapplied. Second, 10,000 bootstrap replicates in TRANSMIT were used todetermine empirical probabilities. The latter approach is particularlyconservative, as it randomly samples a single affected individual foreach family.

The chromosome 4p region was broken into two segments to look for novelcandidate genes: (1) 29-34 cM and (2) 39-43 cM. First, public databaseswere searched for genes in the region near 31-34 cM (corresponding to19-22 mB on the reference assembly), and candidate genes wereidentified. TDT analysis was performed on these genes.

In the first segment (29-34 cM), one of the candidate genes, Kv channelinteracting protein 4 (KCNIP4), was positive (see Table 4).

TABLE 4 TDT Analysis of KCNIP4 SNPs and Haplotypes P values SNPs/HapsSZ + SZ + rs6447982 - rs10016449 SZ SPD SPD + SD Global (3 df) 0.0680.18 0.19 A - T haplotype over-transmitted (1 df) 0.03 0.18 0.25 A - Chaplotype under-transmitted (1 df) 0.16 0.15 0.22 rs#: is the universalSNP identifier used by NCBI (e.g. rs12641357 will return an unique SNPin the human genome sequence). The P values shown are for TDT analysis.Values less than 0.05 are nominally significant and those less than 0.01are highly significant. sz = disease definition is schizophrenia sz + sd= broader disease definition including schizotypal personality disordersz + sd + spd = broadest disease definition including schizoaffectivedisorder

TABLE 5 Bootstrap Replication Analysis of KCNIP4 SNPs and HaplotypesSNPs/Haps SZ + SZ + rs6447982 - rs10016449 SZ SPD SPD + SD Global (3 df)0.023 0.110 0.16 A - T haplotype over-transmitted (1 df) 0.016 0.1300.24 A - C haplotype under-transmitted (1 df) 0.05 0.081 0.16

Table 5 shows the results of bootstrap replication analyses (10,000computer simulations; for selected SNPs and haplotypes only). This is amore conservative way of estimating P values. Low P values by thisprocedure are more likely to be real. The “maximum” values indicate thatmost significant values obtained for any particular haplotype (whichcould be “protective” or “susceptible”). The “global” values indicatethat, as a group, the haplotypes are skewed in their transmission toaffected offspring. For single SNPs the maximum and global scorestheoretically should be equal. For haplotypes involving two or moreSNPs, global values are often more significant, because they reflect thecombined contributions from two or more haplotypes.

The marker D4s391 is at 43.5 cM on the map described above (placed alsoat about 43 to 44 cM on reference maps and corresponding to 27.2 mB onthe reference assembly). The region from 39-43cM corresponds to 24 to 27mB on the reference assembly.

In the second segment (39-43 cM), phosphatidylinositol 4-kinase type 2beta (PI4K2B), at 24.8-24.9 mBase, was positive. The results are shownin Tables 6-7.

TABLE 6 TDT Analysis of PI4K2B SNPs and Haplotypes P values SNPs/HapsSZ + SZ + rs313548 SZ SPD SPD + SD Global (1 df) 0.036 0.018 0.019 Callele over-transmitted (1 df) 0.036 0.018 0.019 rs#: is the universalSNP identifier used by NCBI. The P values shown are for TDT analysis.Values less than 0.05 are nominally significant and those less than 0.01are highly significant. sz = disease definition is schizophrenia sz + sd= broader disease definition including schizotypal personality disordersz + sd + spd = broadest disease definition including schizoaffectivedisorder

TABLE 7 Bootstrap Replication Analysis of PI4K2B SNPs and HaplotypesSNPs/Haps SZ + SZ + rs313548 SZ SPD SPD + SD Global (1 df) 0.031 0.020.042 C allele over-transmitted (1 df) 0.031 0.02 0.042

Thus, SNPs/Haps in the genes KCNIP and PI4K2B on chromosome 4p areassociated with an increased risk of developing SZ, SPD, or SD.

Example 4 Chromosome 22q Detailed QTL Linkage Analysis for MMPI-Scales

The area on chromosome 22q13 was also explored by testing other basicclinical scales and subscales in this region. Only one other basicclinical scale—hypochondriasis (Hathaway and McKinley (1989),supra)—showed elevation in the 22q-ter region with a maximum t=3.05(P=1.2×10⁻³) at our most distal marker, D11S1743, at 64.7 cM andphysically about 10 kb proximal to the coding regions of the ARSA gene(Brennan et al., (2000), supra). The results are shown in Table 8.

TABLE 8 MMPI-2 QTL linkages on Chromosome 22q for Clinical ScalesLinkage Peak cM MMPI-2 Scale t value P value 63   Schizophrenia(schizotypy) 3.83 0.00006 52-53 Schizophrenia (schizotypy) 3.34 0.0004264.7 hypochondriasis 3.05 0.0012 52-54 hypochondriasis 2.81 0.0024

As before, log-transformation had only a small effect. It did not changethe location of the peak at 63 cM, but the t value decrease slightly(t=3.63 vs. 3.83). Similarly, for the secondary peak at 52 cM, theposition did not move, but the t value changed slightly, this time beingsomewhat higher for the log-transformed scale (t=3.55 vs. 3.34). As wasseen for chromosome 4, the results for the log-transformed scoresindicate that the contribution/influence of extreme scores on theselinkage results is small.

Example 5 Chromosome 22q Gene Confirmation by TDT in Clinical Samples

TDT analysis was performed on the identified region of 22q as describedabove in Example 3. This region was broken into two segments: (1) 52-53cM and (2) 63-65 cM, to look for novel candidate genes.

First, public databases were searched for genes in these regions, andcandidate genes were identified for the 52-53 cM region. Of thosecandidates, CERK (45.46-45.51 mBase, known as “FLJ23239”, adiacylglycerol kinase gene of then unknown function), was positive (seeTables 9-10).

TABLE 9 TDT Analysis of CERK SNPs and Haplotypes P values SZ + SZ +SNPs/Haps SZ SPD SPD + SD rs1548977 Global (1 df) 0.045 >0.05 >0.05 Aallele over-transmitted (1 df) 0.045 >0.05 >0.05 rs135667 - rs1548977Global (3 df) 0.0016 0.017 0.011 G - A haplotype over-transmitted (1 df)0.0085 0.055 0.029 G - G haplotype under-transmitted (1 df) 0.0036 0.090.021 rs135678 - rs135693 Global (3 df) 0.14 0.024 0.018 T - C haplotypeover-transmitted (1 df) 0.069 0.024 0.013 rs#: is the universal SNPidentifier used by NCBI. The P values shown are for TDT analysis. Valuesless than 0.05 are nominally significant and those less than 0.01 arehighly significant. sz = disease definition is schizophrenia sz + sd =broader disease definition including schizotypal personality disordersz + sd + spd = broadest disease definition including schizoaffectivedisorder

TABLE 10 Bootstrap Replication Analysis of CERK SNPs and HaplotypesBootstrap Replication SZ + SZ + SNPs/Haps SZ SPD SPD + SD rs1548977Global (1 df) 0.042 >0.05 >0.05 A allele over-transmitted (1 df)0.042 >0.05 >0.05 rs135667 - rs1548977 Global (3 df) 0.0098 0.053 0.064G - A haplotype over-transmitted (1 df) 0.0038 0.039 0.016 G - Ghaplotype under-transmitted (1 df) 0.0055 0.053 0.018 rs135678 -rs135693 Global (3 df) 0.1 0.02 0.02 T - C haplotype over-transmitted (1df) 0.11 0.041 0.024

Table 10 shows the results of bootstrap replication analyses for thepositive SNPs and haplotypes identified in CERK, performed as describedabove in Example 3.

Public database searching for candidate genes in the second region,63-65 cM, identified SH3 and multiple ankyrin repeat domains 3 (SHANK3,at 49.46-49.52 mBase). The results of TDT analysis of SHANK3 SNPs andHaplotypes are shown in Tables 11-12.

TABLE 11 TDT Analysis of SHANK3 SNPs and Haplotypes P values SZ + SZ +SNPs SZ SPD SPD + SD rs9616816 Global (1 df) 0.0095 0.0051 0.0011 Aallele over-transmitted (1 df) 0.0095 0.0051 0.0011 rs713692 - rs9616816Global (3 df) 0.10 0.06 0.02 C - A haplotype over-transmitted (1 df)0.06 0.03 0.015 C - G haplotype under-transmitted (1 df) 0.12 0.19 0.09rs9616915 - rs9616816 Global (3 df) 0.028 0.0055 0.0017 T - A haplotypeover-transmitted (1 df) 0.087 0.058 0.05 C - G haplotypeunder-transmitted (1 df) 0.013 0.0014 0.001 rs9616816-rs739365 Global (3df) 0.066 0.03 0.084 A - C haplotype over-transmitted (1 df) 0.18 0.0690.024 G - C haplotype under-transmitted (1 df) 0.021 0.01 0.0072rs9616816-rs6010063 Global (3 df) 0.0089 0.017 0.0033 A - A haplotypeover-transmitted (1 df) 0.006 0.016 0.0033 G - G haplotypeunder-transmitted (1 df) 0.021 0.055 0.03 rs713692 - rs 756638 Global (3df) 0.37 0.034 0.014 C - A haplotype under-transmitted (1 df) 0.140.0063 0.0015 rs#: is the universal SNP identifier used by NCBI. The Pvalues shown are for TDT analysis. Values less than 0.05 are nominallysignificant and those less than 0.01 are highly significant. sz =disease definition is schizophrenia sz + sd = broader disease definitionincluding schizotypal personality disorder sz + sd + spd = broadestdisease definition including schizoaffective disorder

TABLE 12 Bootstrap Replication Analysis of SHANK3 SNPs and HaplotypesBootstrap Replication SZ + SZ + SNPs SZ SPD SPD + SD rs9616816 Global (1df) 0.042 0.0023 0.0003 A allele over-transmitted (1 df) 0.042 0.00230.0003 rs713692 - rs9616816 Global (3 df) 0.08 0.12 0.051 C - Ahaplotype over-transmitted (1 df) 0.03 0.068 0.035 C - G haplotypeunder-transmitted (1 df) 0.13 0.23 0.120 rs9616915 - rs9616816 Global (3df) 0.040 0.011 0.0037 T - A haplotype over-transmitted (1 df) 0.0210.032 0.027 C - G haplotype under-transmitted (1 df) 0.0095 0.00040.0004 rs9616816-rs739365 Global (3 df) 0.0034 0.011 0.0025 A - Chaplotype over-transmitted (1 df) 0.068 0.0042 0.0001 G - C haplotypeunder-transmitted (1 df) 0.0004 0.0002 0.00003 rs9616816-rs6010063Global (3 df) 0.0001 0.0031 0.0014 A - A haplotype over-transmitted (1df) <0.0001 0.0033 0.0002 G - G haplotype under-transmitted (1 df)0.0032 0.031 0.0037 rs713692 - rs 756638 Global (3 df) ND ND ND C - Ahaplotype under-transmitted (1 df) ND ND ND

Table 12 shows the results of bootstrap replication analyses for thepositive SNPs and haplotypes identified in SHANK3, performed asdescribed above in Example 3.

Example 6 Exemplary SNPs within 1 LDU of Reference SNPs

Public database searches were used to identify exemplary SNPs within 1LDU of the reference SNPs described herein. (From NCBI B36 assembly,dbSNP b126)

KCNP4

SNPs within 1 LDU of marker rs6447982 in European populations include:rs10031524, rs9995697, rs3764964, rs3764965, rs3764966, rs3764967,rs12331966, rs10017693, rs7681691, rs7688592, rs2052775, rs1985322,rs10022322, rs7655154, rs6811030, rs2288308, rs12640448, rs2162413,rs2114474, rs6811505, rs6831295, rs6831516, rs6447975, rs6447976,rs6447978, rs12644782, rs10084802, rs2322688, rs7689421, rs3816874,rs990206, rs7694208, rs6817475; in African populations include:rs10031524, rs10024002, rs9995697, rs7666288, rs3764964, rs3764965,rs3764966, rs12331966, rs10017693, rs7681691, rs7688592, rs2052775,rs10022322, rs7655154, rs9291412, rs6811030, rs2288308, rs2114474,rs6811505, rs6831516, rs6447975, rs6447976, rs6447978, rs12644782,rs10084802, rs2322688, rs7689421, rs3816874, rs990206, rs7694208; inChinese populations include: rs10024002, rs9995697, rs3764964,rs3764965, rs3764966, rs3764967, rs12331966, rs10017693, rs7681691,rs2052775, rs10022322, rs7655154, rs6811030, rs2288308, rs12640448,rs2162413, rs6811505, rs6831295, rs6831516, rs6447975, rs6447976,rs6447978, rs12644782, rs10084802, rs2322688, rs990206, rs10938804,rs12641748; and in Japanese populations include: rs2162413, rs6831295,rs6831516, rs6447975, rs6447978, rs10084802, rs12641748.

SNPs within 1 LDU of marker rs10016449 in European populations include:rs16869961, rs16869962, rs1491363, rs1491364, rs923672, rs13118003,rs1546065, rs12331024, rs13149493, rs6843196, rs9998730, rs1907497,rs13130253, rs16869987, rs16869989, rs1940825, rs7695244, rs7695774,rs10002199; in African populations include: rs13129008, rs3857162,rs1491363, rs1546065, rs6843196, rs9998730, rs1907497, rs16869987,rs1940825, rs7695244; in Chinese populations include: rs16869962,rs1491363, rs13118003, rs16869987, rs16869989, rs10002199; and inJapanese populations include: rs16869962, rs1491363, rs16869987,rs16869989, rs10002199.

PI4K2B

SNPs within 1 LDU of marker rs313548 in European populations include:rs10939041, rs1909475, rs12505283, rs7682177, rs10939043, rs10517063,rs313568, rs313577, rs11940059, rs313567, rs3106321, rs6834255,rs12649921; in African populations include: rs10939047, rs1909475; inChinese populations include: rs10939041, rs1909475, rs10517063,rs313568, rs313577, rs313567, rs3106321, rs6834255, rs12649921; and inJapanese populations include: rs313567, rs3106321, rs6834255,rs12649921, rs10517063, rs313568.

CERK

SNPs within 1 LDU of marker rs15478977 in European populations include:rs1548978, rs5769125, rs1861739, rs5769126, rs710123, rs809652,rs17221476, rs801643, rs5769118, rs4823873, rs12628356, rs4642050,rs738726, rs2080581; in African populations include: rs5769118,rs4823873, rs12628356, rs4642050, rs2080581; in Chinese populationsinclude: rs1548978, rs5769125, rs1861739, rs5769126, rs9626899,rs5769118, rs4823873, rs12628356, rs4642050, rs738726, rs2080581; and inJapanese populations include: rs1548978, rs5769125, rs1861739,rs5769126, rs809652, rs17221476, rs801646, rs9626899, rs5769118,rs4823873, rs12628356, rs4642050, rs738726, rs2080581.

SNPs within 1 LDU of marker rs135667 in European populations include:rs135677, rs801709, rs135678, rs78424, rs135686; in African populationsinclude: rs135668, rs801712, rs135677, rs801709, rs135678, rs885792,rs2542026, rs801724, rs801720, rs2542014; in Chinese populationsinclude: rs135668, rs135677, rs801709, rs135678, rs135680, rs135681,rs135686; and in Japanese populations include: rs801709, rs135678,rs135680, rs135681, rs135686.

SNPs within 1 LDU of marker rs135678 in European populations include:rs78424, rs135686, rs801719, rs135668, rs135677, rs801709; in Africanpopulations include: rs885792, rs2542026, rs801724, rs801720, rs2542014,rs135667, rs135668, rs801712, rs135677, rs801709; in Chinese populationsinclude: rs135680, rs135681, rs135686, rs135698, rs135667, rs135668,rs135677, rs801709; and in Japanese populations include: rs135680,rs135681, rs135686, rs135667, rs135668, rs135677, rs801709.

SNPs within 1 LDU of marker rs135693 in European populations include:rs135694, rs135695, rs85598, rs135697, rs135698, rs135688; in Africanpopulations include: rs135694, rs135695, rs135697, rs5769101, rs2076710,rs2542038, rs2542037, rs6008944, rs737136, rs885792, rs2748341,rs2748343, rs2542026, rs801724, rs2748348, rs801720, rs801719,rs2542014, rs5769083, rs801715, rs135676, rs78424, rs135688; in Chinesepopulations include: rs2076710, rs2542038, rs2542037, rs6008944,rs737136, rs885792, rs2748341, rs2748343, rs2542026, rs801724,rs2748348, rs801720, rs801719, rs2542014, rs5769083, rs801715, rs135676,rs78424, rs135688; and in Japanese populations include: rs135694,rs135695, rs135697, rs5769101, rs2748343, rs2542026, rs801724,rs2748348, rs801720, rs801719, rs2542014, rs5769083, rs801715, rs135676,rs78424, rs135688.

SHANK3

SNPs within 1 LDU of marker rs739365 in European populations include:rs8135777, rs5770820.

SNPs within 1 LDU of marker rs9616816 in European populations include:rs2341009, rs1001469; in African populations include: rs9616915,rs7284093; in Chinese populations include: rs7284093, rs2341009,rs8135777; and in Japanese populations include: rs7284093, rs2341009,rs8135777.

SNPs within 1 LDU of marker rs713692 in European populations includers10854884; in African populations include: rs962185; rs9616812; and inJapanese populations include: rs9616913, rs9616915, rs5770819,rs6009951, rs601006, rs4040041, rs10854884, rs8138460, rs9616906,rs9616812.

SNPs within 1 LDU of marker rs9616915 in European populations include:rs10854884, rs8138460, rs9616906, rs9616812, rs9628185, rs9616913; inAfrican populations include: rs8138460, rs9616913; in Chinesepopulations include: rs8138460, rs9616906, rs9616812, rs9628185,rs9616913; and in Japanese populations include: rs10854884, rs8138460,rs9616906, rs9616812, rs9628185, rs9616913.

SNPs within 1 LDU of marker rs6010063 in European populations include:rs6010065; in Chinese populations include: rs6010065, rs81337951; and inJapanese populations include: rs6010065, rs81337951.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method of identifying a human subject's risk of developingschizophrenia, the method comprising: determining a SH3 and multipleankyrin repeat domains 3 gene (SHANK3) haplotype of the subject, whereinthe SHANK3 haplotype comprises alleles of single nucleotide polymorphismrs9616816 and single nucleotide polymorphism rs6010063; and identifyingthe subject as a human subject having an increased risk of developingschizophrenia when the haplotype comprises an “A” allele at said singlenucleotide polymorphism rs9616816 and an “A” allele at said singlenucleotide polymorphism rs6010063, or identifying the subject as a humansubject having a decreased risk of developing schizophrenia when thehaplotype comprises a “G” allele at said single nucleotide polymorphismrs9616816 and a “G” allele at said single nucleotide polymorphismrs6010063.
 2. The method of claim 1, wherein said determining theSHANK3haplotype comprises: obtaining a sample comprising genomic DNAfrom the subject; and determining the identities of the alleles ofsingle nucleotide polymorphism rs9616816and single nucleotidepolymorphism rs6010063.
 3. The method of claim 2, wherein the sample isobtained from the subject by a health care provider.
 4. The method ofclaim 2, wherein the sample is provided by the subject without theassistance of a health care provider.
 5. The method of claim 1, furthercomprising determining the presence or absence of one or more additionalmarkers associated with schizophrenia in the subject.
 6. The method ofclaim 1, wherein the subject has one or more risk factors associatedwith schizophrenia.
 7. The method of claim 6, wherein said one or moreof the risk factors associated with schizophrenia include: the subjecthas a relative with schizophrenia; the subject has eye trackingdysfunction; the subject has deficits in working memory; or the subjecthas mixed-handedness.
 8. The method of claim 6, wherein the risk factorsassociated with schizophrenia include: the subject has one or morerelatives who have or had schizophrenia and said one or more relativesinclude grandparents, parents, uncles, aunts, siblings, or children ofthe subject.
 9. The method of claim 1, wherein the subject is a child, afetus, or an embryo, and one of the relatives of the subject hasschizophrenia.
 10. A method of identifying a human subject's risk ofdeveloping schizophrenia, the method comprising: determining a SH3 andmultiple ankyrin repeat domains 3 gene (SHANK3) haplotype of thesubject, wherein the SHANK3 haplotype comprises an allele of singlenucleotide polymorphism rs9616816; and identifying the subject as ahuman subject having an increased risk of developing schizophrenia whenthe haplotype comprises an “A” allele at said single nucleotidepolymorphism rs9616816.
 11. The method of claim 10, wherein saiddetermining the SHANK3 haplotype comprises: obtaining a samplecomprising genomic DNA from the subject; and determining the identity ofthe allele of single nucleotide polymorphism rs9616816.
 12. The methodof claim 11, wherein the sample is obtained from the subject by a healthcare provider.
 13. The method of claim 11, wherein the sample isprovided by the subject without the assistance of a health careprovider.
 14. The method of claim 10, further comprising determining thepresence or absence of one or more additional markers associated withschizophrenia in the subject.
 15. The method of claim 10, wherein thesubject has one or more risk factors associated with schizophrenia. 16.The method of claim 15, wherein said one or more of the risk factorsassociated with schizophrenia include: the subject has a relative withschizophrenia; the subject has eye tracking dysfunction; the subject hasdeficits in working memory; or the subject has mixed-handedness.
 17. Themethod of claim 15, wherein the risk factors associated withschizophrenia include: the subject has one or more relatives who have orhad schizophrenia and said one or more relatives include grandparents,parents, uncles, aunts, siblings, or children of the subject.
 18. Themethod of claim 10, wherein the subject is a child, a fetus, or anembryo, and one of the relatives of the subject has schizophrenia.